The 3′ IgH Locus Control Region Is Sufficient to Deregulate a c- myc Transgene and Promote Mature B Cell Malignancies with a Predominant Burkitt-Like Phenotype This information is current as of September 29, 2021. Véronique Truffinet, Eric Pinaud, Nadine Cogné, Barbara Petit, Laurence Guglielmi, Michel Cogné and Yves Denizot J Immunol 2007; 179:6033-6042; ; doi: 10.4049/jimmunol.179.9.6033 http://www.jimmunol.org/content/179/9/6033 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2008/03/12/179.9.6033.DC1 Material http://www.jimmunol.org/ References This article cites 36 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/179/9/6033.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The 3؅ IgH Locus Control Region Is Sufficient to Deregulate a c-myc Transgene and Promote Mature B Cell Malignancies with a Predominant Burkitt-Like Phenotype1 Ve´ronique Truffinet,* Eric Pinaud,* Nadine Cogne´,* Barbara Petit,*† Laurence Guglielmi,2* Michel Cogne´,* and Yves Denizot3* Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either ؅ ؅ close to JH or within switch regions and always link c-myc to the 3 IgH locus control region (3 LCR). To test the hypothesis that the 3؅ LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3؅ LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion Downloaded from proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220؉IgM؉IgD؉ with the BL “starry sky” appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3؅ IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo. The Journal of Immunology, 2007, 179: 6033–6042. http://www.jimmunol.org/ ␮ xpression of c-myc is tightly linked to the early G1 phase linked to E were shown to promote B cell malignancies, although of the cell cycle and plays a critical role in cell prolifer- with a pro-B phenotype (5). Whether E␮ was included, mice car- ation, differentiation, metabolism, and apoptosis (1). Bur- rying yeast artificial chromosomes linking the c-myc gene to a 5Ј E 4 ␦ kitt lymphoma (BL) is characterized by specific chromosomal portion of the IgH locus (VH to C ) developed rather immature B translocations involving the c-myc gene and one of the Ig loci (Ig␬, cell malignancies expressing a variable degree of CD43 (6, 7). Ig␭, and IgH) (2). Once translocated and having lost its normal Altogether, 5Ј elements of the locus induced malignancies with control, c-myc is constitutively expressed throughout the cell cycle features of pre-B, immature B, or transitional B cells rather than by guest on September 29, 2021 in B-lineage cells, the oncogene deregulation being considered the mature B cells as in BL. These results suggest that downstream result from juxtaposition to the Ig gene enhancers. In 80% of cases, IgH elements must deregulate c-myc in BL. the translocation involves the IgH locus (2), itself regulated by a Several lymphoid-specific transcriptional enhancers (hs3a, hs1, complex interplay of regulatory elements responsible for tissue- 2, hs3b, and hs4 in the mouse) have been identified within a 3Ј and stage-specific regulation of both transcription and rearrange- regulatory region lying downstream of the IgH locus. The hs4 ments. Early B cell-specific events such as the germline transcrip- distal element is active from the pre-B cell stage and throughout B tion initiated at the DQ52 promoter and the initiation of VDJ cell ontogeny. A larger region with a global “palindromic” struc- recombination are regulated by upstream elements including the ture encompassing the hs1, 2 central enhancer flanked by inverted E␮ enhancer (3, 4). This cis-acting element was the first candidate repeats, including hs3a and hs3b elements, is active at late B cell proposed for c-myc deregulation in BL because c-myc transgenes stages (8). Altogether, the four elements constitute a potent locus control region (LCR) conferring position-independent and copy- dependent expression to transgenes (9). These elements display *Unite´Mixte de Recherche, Centre National de la Recherche Scientifique 6101, Uni- versite´de Limoges, Limoges, France; and †Service d’Anatomie Pathologique, Uni- specific activity during terminal B cell differentiation, with a major versity Hospital, Limoges, France role in class switch recombination (CSR) (10–12). A construct Received for publication May 11, 2007. Accepted for publication August 13, 2007. containing the four 3Ј IgH enhancers confers, when surrounded by The costs of publication of this article were defrayed in part by the payment of page insulators, an expression from pre-B cells to mature B cells that is charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. strictly confined to the B cell lineage (13). In BL, IgH locus breakpoints are located either in the VDJ (en- 1 This work was supported by grants from Ligue Nationale contre le Cancer, Conseil Re´gional du Limousin, and “Lions Club de la Corre`ze, Zone 33 district 103 Sud.” demic BL) or the switch region (sporadic BL). When upstream of 2 Current address: Institut National de la Sante´et de la Recherche Me´dicale Unite´ E␮, the chromosomal breakage may result from an abnormal so- 454, Centre Hospitalier de l’Universite´Arnaud de Villeneuve, Montpellier, France. matic hypermutation event in early germinal center B cells. Break- 3 Address correspondence and reprint requests to Dr. Yves Denizot, Faculte´Me´- points located within switch regions are rather initiated by an er- decine, Laboratoire d’Immunologie, Unite´Mixte de Recherche, Centre National de la Recherche Scientifique 6101, 2 rue Dr. Marcland, 87025, Limoges, France. E-mail roneous class switching and link c-myc to a downstream portion of address: [email protected] the locus lacking E␮ (2). By contrast, 3Ј IgH enhancers are always 4 Abbreviations used in this paper: BL, Burkitt lymphoma; LCR, locus control region; conserved on the c-myc-translocated chromosome and constitute CSR, class switch recombination; ES, embryonic stem; wt, wild type; WBC, white candidates for oncogene deregulation. blood cell. In 20% of cases, c-myc translocation involved the Ig L chain Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 (Ig␬ and Ig␭) loci. A BL-like model has been previously generated www.jimmunol.org 6034 c-myc-3Ј LCR AND BL by using the c-myc gene fused to elements of the ␭ L chain locus ␮l. Quantitative PCR was performed in duplicate by using TaqMan assay (14). However, in 80% of cases, c-myc translocation involved the reagents and analyzed on an ABI Prism 7000 system (Applied Biosys- IgH locus. For now, two mouse models have closely mimicked tems). Product reference for c-myc was Mm00487803-m1 and for the 18S RNA used for normalization of gene expression levels was Hs99999901-s1 c-myc translocation into the IgH locus. In one case, a c-myc in- (Applied Biosystems). Primers and probe for the P1 promoter were: 5Ј- sertion downstream of JH imitates the translocation observed in GAGGGATCCTGAGTCGCAGTAT-3Ј,5Ј-CTCTGCACACACGGCTCTT endemic BL. Aged animals develop B cell and plasma cell neo- C-3Ј and 5Ј-(6-FAM)TCTCCTGCCTGATAAACAACAACTTGGGATG-3Ј. plasms resulting from c-myc activation (15, 16). Deregulation of Flow cytometry analysis c-myc cannot be attributed to any precise cis-responsive elements because the transgene is integrated in the whole IgH locus chro- Single-cell suspensions from thymus, spleen, liver, and bone marrow were matin environment. In the second model, a truncated 3Ј IgH cas- labeled with various Abs (Southern Biotechnology Associates): anti-B220- conjugated with Spectral Red (PC5), anti-CD19, anti-CD117, anti-IgM, sette (lacking hs3a) was integrated upstream of the c-myc-coding and anti-IgD conjugated with PE. Control experiments included irrelevant region on mouse chromosome 15 and induced B cell lymphomas isotype-matched Abs conjugated with PC5 or PE. Cells were analyzed on in aged animals (17). This latter model demonstrated the important a Coulter XL apparatus (Beckman Coulter). role of the 3Ј IgH LCR on c-myc deregulation, although it is not excluded that the knockin might also have disrupted upstream Proliferation and cell cycle analysis and/or downstream c-myc regulatory elements (18, 19).