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Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease

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Immunoglobulin transcription is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD). We recently demonstrated that defective immunoglobulin promoter transcription correlates with the down-regulation of the B-cell
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  doi:10.1182/blood.V99.8.30602002 99: 3060-3062 Mathas, Claus Scheidereit, Thomas Wirth, Harald Stein and Bernd DörkenFranziska Jundt, Katharina Kley, Ioannis Anagnostopoulos, Kristina Schulze Pröbsting, Axel Greiner, Stephan  diseasetranscription in Hodgkin and Reed-Sternberg cells of classical Hodgkin Loss of PU.1 expression is associated with defective immunoglobulin   http://bloodjournal.hematologylibrary.org/content/99/8/3060.full.html Updated information and services can be found at: (4217 articles)Neoplasia  (1653 articles)Brief Reports  Articles on similar topics can be found in the following Blood collections  http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at:  http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at:  http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Copyright 2011 by The American Society of Hematology; all rights reserved.American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the  For personal use only.by guest on June 5, 2013. bloodjournal.hematologylibrary.orgFrom   Brief report LossofPU.1expressionisassociatedwithdefectiveimmunoglobulintranscriptioninHodgkinandReed-SternbergcellsofclassicalHodgkindisease Franziska Jundt, Katharina Kley, IoannisAnagnostopoulos, Kristina Schulze Pro¨bsting, Axel Greiner, Stephan Mathas,Claus Scheidereit, Thomas Wirth, Harald Stein, and Bernd Do¨rken Immunoglobulintranscriptionisimpairedin Hodgkin and Reed-Sternberg (HRS)cells of classical Hodgkin disease (cHD).We recently demonstrated that defectiveimmunoglobulin promoter transcriptioncorrelateswiththedown-regulationoftheB-cell transcription factors Oct2 andBOB.1/OBF.1. These results prompted usto investigate whether immunoglobulinenhancer activity is also impaired in HRScells and whether as yet unidentified fac-torscouldbenecessaryforimmunoglobu-lin enhancer activity in HRS cells of cHD.Here we analyzed 30 cases of cHD forexpressionoftheEtsfamilymemberPU.1that is known to collaborate with multipletranscription factors and to regulate ex-pression of immunoglobulin genes. Weshow that PU.1 is not expressed in pri-mary and cultured HRS cells. Reintroduc-tion of PU.1 and Oct2 in cultured HRScells restored the activity of cotrans-duced immunoglobulin enhancer con-structs. Our study identifies PU.1 defi-ciency as a recurrent defect in HRS cellsthat might contribute to their impairmentof immunoglobulin transcription. (Blood.2002;99:3060-3062) ©  2002 by TheAmerican Society of Hematology Introduction Absent immunoglobulin expression despite the presence of clonalimmunoglobulin gene rearrangements is a hallmark of tumor cellsof classical Hodgkin disease (cHD). 1 Recently, we showed thatcrippling mutations in the immunoglobulin coding region and inthe octamer motif of the immunoglobulin promoter are rare eventsin cHD. 1 We further demonstrated that defective immunoglobulinpromoter transcription is most likely due to the down-regulatedexpression of B-cell–specific transcription factors such as Oct2 andBOB.1/OBF.1. 1-3 In this study, we investigated whether immuno-globulin enhancer activity is also impaired in Hodgkin andReed-Sternberg (HRS) cells and whether as yet unidentified factorscould be necessary for immunoglobulin enhancer activity in HRScells of cHD. We, therefore, analyzed the expression of the Etsfamily transcription factor PU.1/SPI-1 that is known to essentiallycontribute to the regulation of immunoglobulin-  , -  , and -   geneexpression. 4 PU.1 is a tissue-restricted transcriptional regulator within thehematopoietic system that is essential for the development of bothB cells and macrophages. 4-9 Furthermore, several DNA-bindingstudies revealed that PU.1 binds to a large number of promoters andregulates the expression of genes that are required for terminaldifferentiation of B cells. 9,10 The list includes genes encodingimmunoglobulin light (   or   ) and heavy chain genes, RAG-1,immunoglobulin    (Ig-  ), Ig-  , Vpre-B,   5, CD19, andJ-chain. 5,10-14 Thereby, PU.1 requires interactions with other tran-scription factors to regulate transcription and plays an architecturalrole in forming higher-order complexes. 15-17 Here we show that PU.1 is not detectable in cultured andprimary HRS cells of cHD. We demonstrate that cotransfection of PU.1 and Oct2 in transient transfection studies activates immuno-globulin heavy chain (IgH) intronic enhancer expression in cul-tured HRS cells. Together with the absence of Oct2 and BOB.1/ OBF.1, PU.1 deficiency is an important recurrent defect of HRScells probably contributing to defective immunoglobulin geneexpression in cHD. Study design Formalin-fixed and paraffin-embedded lymph node specimens from 30patients with the diagnosis of cHD of nodular sclerosis or mixed cellularitysubtype were retrieved from the files of the Reference Center for LymphNode Pathology and Hematopathology at the Institute of Pathology,Klinikum Benjamin Franklin, Free University of Berlin, Berlin, Germany.Activated lymphoid tissue from palatinal tonsils served as control. Tissuesections (4   m) were deparaffinized and subjected to a brief high-temperature pretreatment with the use of a pressure cooker and 0.01 Mcitrate buffer pH 6.0. The sections were incubated with the anti-PU.1monoclonal antibody (clone G148-74 purchased from Becton DickinsonBiosciences, Heidelberg, Germany). Bound antibody was visualized byusing the EnVision Kit (DAKO, Glostrup, Denmark).Cell extracts were prepared and quantitated as described. 18 The primaryantibodies were anti-PU.1 (Santa Cruz, CA) and anti-Spi-B (M.C. Simon,Howard Hughes Medical Institute, Philadelphia, PA) antibodies.Electroporation was performed as recently described. 2,3 The luciferasereporter plasmids driven by conalbumin promoter with or without the  MluI-HpaI   fragment of the human IgH gene intronic enhancer (pEcona-Lucand pcona-Luc) were provided by Dr T. Watanabe (Kyusyu University,Fukuoka, Japan 19 ). Murine PU.1 complementary DNAwas provided by DrD. Tenen (Harvard Medical School, Boston, MA) and cloned in apCDNA-3.1 expression plasmid (Invitrogen, Groningen, The Netherlands). From the Charite´, Robert-Ro¨ssle-Klinik, Humboldt University of Berlin,Germany; Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany;Institute of Pathology, Klinikum Benjamin Franklin, Free University of Berlin,Germany; Institute of Pathology, University of Wu¨rzburg, Germany;Department of Physiological Chemistry, University of Ulm, GermanySubmitted September 4, 2001; accepted November 30, 2001.Supported by the Deutsche Forschungsgemeinschaft through JU 426/1-1. Reprints: FranziskaJundt,Charite´,Robert-Ro¨ssle-Klinik,HumboldtUniversityof Berlin, D-13125 Berlin, Germany; e-mail: fjundt@mdc-berlin.de.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’in accordance with 18 U.S.C. section 1734. © 2002 by TheAmerican Society of Hematology3060 BLOOD, 15APRIL 2002    VOLUME 99, NUMBER 8  For personal use only.by guest on June 5, 2013. bloodjournal.hematologylibrary.orgFrom   Results and discussion In this study we investigated the role of the B-cell transcriptionfactor and Ets family member PU.1 in defective immunoglobulingene expression in HRS cells of cHD. For this purpose we analyzed30 cases of cHD for expression of PU.1 by immunohistochemistry.InallcasesPU.1expressionwastotallyabsentfromHRScells,whereaswe found, as expected, PU.1 protein levels in reactive B cells,granulocytes, and macrophages surrounding HRS cells (Figure 1B).PU.1expressionwasfurtherdetectedinthevariousB-cellcompart-ments of reactive lymphoid tissues that served as a positive control(Figure 1A). In contrast to our analysis of the transcription factorOct2 and its coactivator BOB.1/OBF.1, that were partially presentat low levels in 9.4% and 25% of cases of cHD respectively, 2 PU.1expression was not detectable in any case. PU.1 deficiency,therefore, is a recurrent defect of a B-cell transcription factor inHRS cells of cHD, underlining our hypothesis that absent immuno-globulin expression in cHD is probably due to defects in thetranscriptional machinery. These data are in accordance with ourrecent results, showing that neither crippling mutations within theV region gene nor in the octamer motif of the immunoglobulinpromoter or other regulatory sequences can be the general cause forthe defective immunoglobulin transcription in cHD. 1-3 To verify PU.1 deficiency in cultured HRS cells, we performedWestern blot analysis by using PU.1-specific antibodies (Figure2A). As expected, all Hodgkin cell lines (lanes 1-6) did not showPU.1 protein levels in contrast to other B-cell–derived non-Hodgkin cell lines (lanes 7-9) and CD19  B cells (lane 10).Correlating with the absence of PU.1 protein levels, we did notdetect any PU.1-specific transcripts in the Hodgkin cell lines byNorthern blot analysis (data not shown). Whereas PU.1 messengerRNA was readily detectable in the non-Hodgkin cell lines tested(data not shown).However, Spi-B, another member of the Ets family of DNA-binding proteins, 20,21 was expressed in several Hodgkin andnon-Hodgkin cell lines (Figure 2A; lanes 1, 2, 5-8) and in CD19  Bcells (lane 10). Our data indicate that in contrast to the Ets familymember Spi-B, PU.1 is not expressed in cultured HRS cells. Alltested Hodgkin cell lines, therefore, resemble tumor cells in vivowith respect to PU.1 expression. The correspondence between ourin vitro and in vivo data is particularly important, because recentstudies of our group showed that there might be discrepant resultsstudying B-cell transcription factors in cultured and primary HRScells. High expression of Oct2, shown in cultured HRS cells byelectromobility shift assays, could subsequently neither be detectedin primary nor in cultured tumor cells by our very sensitive in situhybridization analysis. 2,22 This discrepancy could possibly beexplained by cell culture conditions that might have transitorilyinduced Oct2 levels in the Hodgkin cell lines.There is abundant evidence that PU.1 is important for heavy andlight chain immunoglobulin gene transcription. 10-14 PU.1 stimu-lates, for example, the activity of the immunoglobulin-   enhancerby playing an architectural role in the assembly of a higher-orderprotein-DNA complex. 17 Thereby binding of phosphorylated PU.1to its target sequence in the 3   enhancer results in the cooperative Figure 1. Expression pattern of PU.1 in reactivelymphoidtissueandinclassicalHodgkindisease. (A)Uniform nuclear expression of PU.1 by germinal centerand follicular mantle B cells in tonsillar lymphoid tissue.(B) Hodgkin and Reed-Sternberg cells show absent PU.1expression, whereas small B cells and occasional histio-cytes express the protein in the nuclei (immunostainsusing a PU.1 monoclonal antibody and the EnVisionmethod using diaminobenzidine as chromogen, brownreaction product). Isotype-matched irrelevant antibodiesdid not show positive staining patterns on the sametissues. Original magnificationA,  100; B,  250. Figure2.PU.1expressioninHodgkincelllinesandtransientcotransfectionsofthe Hodgkin cell lines L428 and KM-H2 with immunoglobulin enhancer con-structs.  (A) Western blot analysis of cell lysate proteins from Hodgkin (lanes 1-6),non-Hodgkin(Burkittlymphoma:Daudi,Namalwa;pre-Bcellline:Reh;lanes7-9)celllines and mature CD19  B cells (B cells; lane 10), showing bands of PU.1 and Spi-B.Left margin, size markers in kilodalton. L428 (B) and KM-H2 (C) cells weretransfected with luciferase reporter plasmids driven by conalbumin promoter with orwithout the  MluI-HpaI   fragment of the human IgH gene intronic enhancer (pEconaand pcona). Expression vectors for PU.1, Oct2, and BOB/OBF.1 were cotransfectedas indicated. Relative luciferase activity is shown and transfections with emptyexpression vectors were arbitrarily set to 1. PU.1 EXPRESSION IN HODGKIN DISEASE 3061BLOOD, 15APRIL 2002    VOLUME 99, NUMBER 8  For personal use only.by guest on June 5, 2013. bloodjournal.hematologylibrary.orgFrom   recruitment of additional transcription factors such as Pip, E2A,and AP-1 to adjacent DNA sites. 17 However, none of the enhancerbinding proteins alone can significantly activate the 3   enhancer. 17 To investigate whether PU.1 can contribute to immunoglobulinenhancer activity in cultured HRS cells, we performed cotransfec-tion experiments in which we reintroduced PU.1 and Oct2 as wellas the Oct2 coactivator BOB.1/OBF.1 into L428 and KM-H2 cells(Figure 2B,C). By using luciferase reporter constructs driven byconalbumin promoter with the human IgH intronic enhancer, weshowed that in L428 and KM-H2 cells PU.1, Oct2, and BOB.1/ OBF.1 alone only slightly activated reporter constructs. However,reintroduction of PU.1 together with Oct2 (L428 cells, Figure 2B;KM-H2 cells, Figure 2C) significantly enhanced reporter activity.This activity was identical to that obtained in the Burkitt lymphomacell lines Daudi and Namalwa (data not shown), expressingendogenous levels of these factors (Figure 2Aand data not shown).Our data indicate that in HRS cells PU.1 can cooperate with Oct2 inactivation of the human IgH gene intronic enhancer. These data arein line with a previous report, suggesting that PU.1 plays a generalrole for the activation of immunoglobulin gene transcription. 10 According to these data PU.1 can bind to pyrimidine-rich motifsthat are functionally important, in particular for those regulatoryelements that have only imperfect octamer sites. PU.1 and Oct2bindconcomitantlytotheseelementsandtherebyactivateimmuno-globulin gene transcription. 10 Because there are binding sites in theIgH intronic enhancer for PU.1 and Oct2, it seems conceivable thatthey regulate enhancer activity in our reporter constructs.In our recent study we used reporter constructs with thewild-type or mutated immunoglobulin promoter octamer motif. 2 We demonstrated a high transcriptional activation of the immuno-globulin promoter construct after cotransfection of Oct2 and/or itscoactivator BOB.1/OBF.1 in L428, KM-H2, and L1236 cells. 2,3 Weconclude from our recent and present results that in HRS cellsdependent on the immunoglobulin promoter or enhancer context,Oct2, BOB.1/OBF.1, and PU.1 can contribute to their transcrip-tional activation.In summary, our data suggest that the loss of expression of several important B-cell transcription factors such as Oct2, BOB.1/ OBF.1, and PU.1 is responsible for the inactivity of immunoglobu-lin promoters and enhancers in HRS cells of cHD. References 1. Marafioti T, Hummel M, Foss H-D, et al. Hodgkinand Reed-Sternberg cells represent an expan-sion of a single clone srcinating from a germinalcenter B-cell with functional immunoglobulin generearrangements but defective immunoglobulintranscription. Blood. 2000;95:1443-1450.2. Stein H, Marafioti T, Foss H-D, et al. Down-regu-lation of BOB.1/OBF.1 and Oct2 in classicalHodgkin disease but not in lymphocyte predomi-nant Hodgkin disease correlates with immuno-globulin transcription. Blood. 2001;97:496-501.3. Theil J, Laumen H, Marafioti T, et al. Defectiveoctamer-dependent transcription is responsiblefor silenced immunoglobulin transcription inReed-Sternberg cells. Blood. 2001;97:3191-3196.4. Lloberas J, Soler C, CeladaA, et al. The key roleof PU.1/SPI-1 in B cells, myeloid cells and macro-phages. Immunol Today. 1999;20:184-189.5. HendersonA, Calame K. Transcriptional regula-tion during B cell development.Annu Rev Immu-nol. 1998;16:163-200.6. Chen H-M, Zhang P, Radomska HS, et al. Oc-tamer binding factors and their coactivator canactivate the murine PU.1 (spi-1) promoter. J BiolChem. 1996;271:15743-15752.7. Klemsz MJ, McKercher SR, CeladaA, Van Bev-eren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to theets oncogene. Cell. 1990;61:113-124.8. McKercher SR, Torbett BE,Anderson KL, et al.Targeted disruption of the PU.1 gene results inmultiple hematopoietic abnormalities. EMBO J.1996:15:5647-5658.9. DeKoter RP, Singh H. Regulation of B lymphocyteand macrophage development by graded expres-sion of PU.1. Science. 2000;288:1439-1441.10. Schwarzenbach H, Newell JW, Matthias P. In-volvement of the Ets family factor PU.1 in the ac-tivation of immunoglobulin promoters. J BiolChem. 1993;270:898-907.11. Kistler B, Pfisterer P, Wirth T. Lymphoid- and my-eloid-specific activity of the PU.1 promoter is de-termined by the combinatorial action of octamerand ets transcription factors. Oncogene. 1995;11:1095-1106.12. Pongubala JMR, Nagulapalli S, Klemesz MJ, etal. PU.1 recruits a second nuclear factor to a siteimportant for immunoglobulin kappa 3   enhanceractivity. Mol Cell Biol. 1992;12:368-378.13. Nelsen B, Tian G, Erman B, et al. Regulation oflymphoid immunoglobulin mu heavy chain geneenhancer by ETS-domain proteins. Science.1993;261:82-86.14. Rivera RR, Stuiver MH, Steenbergen R, Murre C.Ets proteins: new factors that regulate immuno-globulin heavy-chain gene expression. Mol CellBiol. 1993;13:7163-7169.15. Eisenbeis CF, Singh H, Storb U. Pip, a novel IRFfamily member, is a lymphoid-specific, PU.1-de-pendent transcriptional activator. Genes Dev.1995;9:1377-1387.16. Mittru¨cker HW, Matsuyama T, GrossmanA. Re-quirement for the transcription factor LSIRF/IRF4for mature B and T lymphocyte function. Science.1997;275:540-543.17. Pongubala JMR,Atchison ML. PU.1 can partici-pate in an active enhancer complex without itstranscriptional activation domain. Proc NatlAcadSci U SA. 1997;94:127-132.18. Jundt F,Anagnostopoulos I, Bommert K, et al.Hodgkin/Reed-Sternberg cells induce fibroblaststo secrete eotaxin, a potent chemoattractant for Tcells and eosinophils. Blood. 1999;94:2065-2071.19. Wang J, Oketani M, Watanabe T. Positive andnegative regulation of immunoglobulin gene ex-pression by a novel B-cell-specific enhancer ele-ment. Mol Cell Biol. 1991;11:75-84.20. Garrett-Sinha LA, Su GH, Rao S, et al. PU.1 andSpi-B are required for normal B cell receptor-me-diated signal transduction. Immunity. 1999;10:399-408.21. Su GH, Ip HS, Cobb BS, et al. The ets proteinSpi-B is expressed exclusively in B cells and Tcells during development. J Exp Med. 1996;184;203-214.22. Bargou RC, Leng C, Krappmann D, et al. High-level nuclear NF-  B and Oct-2 is a common fea-ture of cultured Hodgkin/Reed-Sternberg cells.Blood. 1996;87:4340-4347. 3062 JUNDTet al BLOOD, 15APRIL 2002    VOLUME 99, NUMBER 8  For personal use only.by guest on June 5, 2013. bloodjournal.hematologylibrary.orgFrom 
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