PU.1 Regulates Positive Regulatory Domain I-Binding Factor 1/Blimp-1 Transcription in Lymphoma Cells This information is current as Shruti Desai, Sophia C. E. Bolick, Michelle Maurin and of September 27, 2021. Kenneth L. Wright J Immunol 2009; 183:5778-5787; Prepublished online 14 October 2009; doi: 10.4049/jimmunol.0901120 http://www.jimmunol.org/content/183/9/5778 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2009/10/13/jimmunol.090112 Material 0.DC1 http://www.jimmunol.org/ References This article cites 48 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/183/9/5778.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology PU.1 Regulates Positive Regulatory Domain I-Binding Factor 1/Blimp-1 Transcription in Lymphoma Cells1 Shruti Desai,2*† Sophia C. E. Bolick,2,3*§ Michelle Maurin,* and Kenneth L. Wright4*†‡§ The human positive regulatory domain I-binding factor 1 (PRDI-BF1) and its murine homolog Blimp-1 promote differentiation of mature B cells into Ab-secreting plasma cells. In contrast, ectopic expression of PRDI-BF1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1, the gene encoding PRDI-BF1. This report establishes that in lymphoma cells stimulation through the BCR rapidly induces endogenous PRDM1 at the level of transcription with minor changes in mRNA stability. The induced PRDM1-encoded protein localizes to its target genes in vivo and suppresses their expression. In vivo genomic footprinting of the PRDM1 promoter in unstimulated lymphoma and myeloma cells reveals multiple common in vivo occupied elements throughout the promoter. Further functional and structural analysis of the promoter reveals that the promoter is preloaded and poised for activation in the B cell lines. The transcription Downloaded from factor PU.1 is shown to be required for the BCR-induced expression of PRDM1 in lymphoma cells and in PU.1-positive myeloma cells. Activation of PRDM1 is associated with loss of the corepressor transducin-like enhancer of split 4 from the PU.1 complex. These findings indicate that PRDM1 is poised for activation in lymphoma cells and therefore may be a potential therapeutic target to inhibit lymphoma cell proliferation and survival. The Journal of Immunology, 2009, 183: 5778–5787. he positive regulatory domain I-binding factor-1 (PRDI- Anti-IgM cross-linking of the BCR has been reported in multi- http://www.jimmunol.org/ BF1),5 encoded by the PRDM1 gene, was originally ple studies to induce apoptosis in lymphoma cells (10–14). This T found to specifically bind the IFN-␤ promoter and sup- response has been correlated with decreased levels of c-myc (6). press IFN-␤ transcription following viral induction (1). Blimp-1, Inducing PRDI-BF1/Blimp-1 expression in lymphoma cells with the murine homolog of PRDI-BF1, was originally described by histone deacetylase inhibitors also decreased expression of the Turner et al. (2) as a transcription factor that could induce the downstream targets c-myc and BSAP (15). More specifically, in- differentiation of B cells. PRDI-BF1/Blimp-1 has since been found troduction of PRDI-BF1/Blimp-1 into lymphoma cells can induce to be required for the differentiation of a B cell to a plasma cell (3). apoptosis, suggesting PRDI-BF1 may be an important mediator of During the differentiation of mature B cells to plasma cells, PRDI- the anti-IgM-mediated apoptotic response (16, 17). However, no BF1 represses multiple genes involved in maintaining the B cell direct link between expression of PRDI-BF1/Blimp-1- and anti- by guest on September 27, 2021 phenotype and in maintaining cellular proliferation, such as CIITA IgM-mediated BCR activation has been described. (4, 5), c-myc (6), and B cell lineage-specific activator protein Recently, PRDM1 expression has been detected in a subset of (BSAP; Ref. 7). Microarray studies have outlined the PRDI-BF1 diffuse large B cell lymphomas (18–20). However, inactivating repression profile and led to the identification of two additional mutations in the PRDM1 coding sequence were described, indi- direct targets, Spi-B and Id3 (8). Additionally, expression of the cating a potential tumor suppressor role for this gene (19, 20). PRDM1 gene has recently been linked to cellular stress and the Similarly, proliferating myeloma cells and myeloma cell lines unfolded protein response in B cells (9). abundantly express the truncated PRDI-BF1 isoform, PRDI-BF1␤, which has impaired function (21). Additionally, Borson et al. (22) demonstrated PRDM1 expression in B cells isolated from my- *H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612; †Cancer Biology Ph.D. Program, ‡Department of Oncologic Sciences, and §Department of eloma patients, whereas normal donors lack expression. The mu- Molecular Medicine, University of South Florida, Tampa, FL 33612 tation status of PRDM1 in these myeloma-derived B cells is as yet Received for publication April 7, 2009. Accepted for publication September 3, 2009. unknown. Together, these findings indicate that PRDI-BF1/ The costs of publication of this article were defrayed in part by the payment of page Blimp-1 may be important to the pathology of various hemopoietic charges. This article must therefore be hereby marked advertisement in accordance malignancies, including lymphoma. with 18 U.S.C. Section 1734 solely to indicate this fact. Very little is known as to the regulation of PRDM1 expression. 1 This work was supported by National Institutes of Health Grants CA114504 and PRDM1 CA080990. Our data now demonstrate that is regulated primarily at the level of transcription both in myeloma cells and in lymphoma 2 These authors contributed equally to the work presented in this report. cells stimulated by cross-linking of the BCR. BCR stimulation 3 Current address: National Institute of Environmental Health Sciences, Laboratory of Molecular Carcinogenesis, 111 TW Alexander Drive, Room C310, Research Triangle leads to rapid increases in newly transcribed PRDM1 RNA levels, Park, NC 27709. whereas mRNA stability is unchanged. Using promoter deletion 4 Address correspondence and reprint requests to Dr. Kenneth L. Wright, H. Lee constructs, we demonstrate several regions of activation in the Moffitt Cancer Center, 12902 Magnolia Drive, MRC-4 East, Tampa, FL 33612. PRDM1 promoter in both lymphoma and myeloma cells. In vivo E-mail address: ken.wright@moffitt.org genomic footprinting demonstrates multiple protein-DNA interac- 5 Abbreviations used in this paper: PRDI-BF1, positive regulatory domain I-binding factor 1; BSAP, B cell lineage-specific activator protein; FWD, forward; REV, re- tions in both lymphoma and myeloma cells. Further analysis of verse; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; UTR, these interactions reveals PU.1 binding is functionally important untranslated region; TLE4, transducin-like enhancer of split 4; CBP, CREB-binding for promoter activity in stimulated lymphoma cells. These findings protein; BCL6, B cell lymphoma 6. demonstrate the PRDM1 promoter is poised for rapid activation in Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 lymphoma cells, which suggests that inducing PRDM1 expression www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901120 The Journal of Immunology 5779 in lymphoma cells may be a viable target to inhibit lymphoma Transfections and luciferase assays progression. Cells were transfected by electroporation using the Gene Pulser II (Bio- Rad). Cells (1 ϫ 107) were pulsed with 250 V at a capacitance of 1070 ␮F. Materials and Methods Transfections for luciferase assays were done with 10 ␮g of luciferase reporter construct and 50 ng of the internal control plasmid pRL-TK. Fire- Cell lines and reagents fly luciferase activity was normalized to Renilla luciferase activity in all The CA46 EBV-negative Burkitt’s lymphoma and RPMI8226 multiple experiments. For small interfering RNA (siRNA) knockdown experiments myeloma cell lines were maintained in RPMI (Invitrogen) supplemented in CA46, cells were transfected with 3 ␮g of control nontargeting siRNA with 10% FBS (HyClone), and 1% penicillin/streptomycin (Invitrogen). or PU.1 siRNA (Dharmacon) for 24 h. Live cells were separated using a Goat anti-human IgM Ab (Southern Biotechnology) was used at 10 ␮g/ml. Ficoll gradient and treated with 10 ␮g of anti-IgM for 24 h. For siRNA Actinomycin D (Sigma-Aldrich) was used at 10 ␮g/ml. knockdown experiments in RPMI8226, cells were transfected with 2 ␮gof control nontargeting siRNA or PU.1 siRNA (Dharmacon) for 48 h. B cell isolation Dimethyl sulfate in vivo footprinting