Increased B Cell Proliferation and Reduced Ig Production in DREAM Transgenic Mice Magali Savignac, Britt Mellström, Anne-Gaëlle Bébin, Juan C. Oliveros, Laurent Delpy, Eric Pinaud and Jose R. Naranjo This information is current as of September 29, 2021. J Immunol 2010; 185:7527-7536; Prepublished online 8 November 2010; doi: 10.4049/jimmunol.1000152 http://www.jimmunol.org/content/185/12/7527 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/11/08/jimmunol.100015 Material 2.DC1

References This article cites 38 articles, 18 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/185/12/7527.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021

• 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Increased B Cell Proliferation and Reduced Ig Production in DREAM Transgenic Mice

Magali Savignac,*,† Britt Mellstro¨m,*,‡ Anne-Gae¨lle Be´bin,x Juan C. Oliveros,* Laurent Delpy,x Eric Pinaud,x and Jose R. Naranjo*,‡

DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum Ig levels. In vitro assays show that reduced Ig secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation, class switch recombination, or Ig transcription. Surprisingly, transgenic B cells show an accelerated entry in cell division. Transcriptomic analysis of transgenic B cells revealed that hyperproliferative B cell response could be correlated with a reduced expression of Klf9, a cell-cycle regulator. Pulse-chase experi- ments demonstrated that the defect in Ig production is associated with reduced translation rather than with increased degradation. Importantly, transgenic B cells showed reduced expression of the Eif4g3 , which encodes a protein related to Downloaded from protein translation. Our results disclose, to our knowledge, a novel function of DREAM in proliferation and Ig synthesis in B lymphocytes. The Journal of Immunology, 2010, 185: 7527–7536.

ntigen exposure activates a profound structural and func- the unfolded protein response (UPR), a global cellular reaction that tional metamorphosis in small resting B lymphocytes controls expression of ER molecular chaperones, protein synthesis A leading to proliferation and differentiation into Ab-se- and degradation, cell cycle progression and apoptosis (4). http://www.jimmunol.org/ creting plasma cells. This complex program involves proliferation, The transcriptional repressor downstream regulatory element generationofmemorycells,isotypeswitching,andaffinitymaturation antagonist modulator (DREAM) is a Ca2+-binding protein that (1, 2). The proliferative phase encompasses multiple rounds of di- binds specifically to downstream regulatory element (DRE) se- vision and is followed by an extensive differentiation process driven quences in DNA (5). The level of nuclear Ca2+, the interaction with by specific changes in (3). During plasma cell dif- other nucleoproteins such as cAMP response element modulator ferentiation, B lymphocytes undergo a dramatic architectural trans- and CREB, and the PI3K pathway regulate the binding of DREAM formation with expansion of the intracellular membrane network, to DRE sites (5–8). Mutation of two key amino acids within any of particularly the rough endoplasmic reticulum (ER), where Igs are the functional EF hands of DREAM results in a protein insensitive by guest on September 29, 2021 synthesized and assembled into functional Abs. Igs mature through to Ca2+ (EFmDREAM) that remains bound to DNA during Ca2+ the Golgi complex and then travel through the exocytic pathway to stimulation (5). Because DREAM binds to DRE sites as a tetramer, be secreted. Increased demands on the secretory machinery trigger EFmDREAM is shown to function as a dominant active mutant in a background of wild-type DREAM in vitro (9) and endogenous *Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas; DREAM in vivo (10, 11). Moreover, DREAM can impair the re- ‡Centro Investigacion Biomedica En Red en Enfermedades NEuroDegenerativas, x cruitment of CREB-binding protein (CBP) by phospho-CREB and Madrid, Spain; †Purpan Hospital, INSERM, U563, Toulouse; and National Center for Scientific Research, University of Limoges UMR 6101, Faculty of Medicine, block CBP-mediated transactivation at cAMP response element Limoges, France sites in a Ca2+-dependent manner allowing cross talk between Received for publication January 20, 2010. Accepted for publication October 6, cAMP and Ca2+ signaling pathways in the nucleus (7). Two 2010. leucine-charged residue rich domains mediate protein–protein This work was supported by grants from the European Community (NoE/512032), interactions of the DREAM protein. The L56V mutation in the Fundacion La Caixa, Ministerio de Ciencia e Innovacion, and the Center for Bio- medical Research in Neurodegenerative Diseases Network (to J.R.N. and B.M.). M.S. leucine-charged residue rich domain of DREAM, which is re- was supported by the Association pour la Recherche sur le Cancer, Marie Curie sponsible for the DREAM–CREB interaction, results in a mutant Individual Fellowship, and a Ramon y Cajal Senior Scientist contract. (LmDREAM) that fails to block CBP-mediated transactivation at The sequences presented in this article have been submitted to the Gene Expression cAMP response element sites. Thus, the dominant active double Omnibus database under accession number GSE16874. mutant of DREAM (EFLmDREAM, hereafter called daDREAM) Address correspondence and reprint requests to Jose R. Naranjo, Centro Nacional de 2+ Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain, Dar- will specifically block Ca -induced de-repression of DREAM win 3, Cantoblanco Campus, 28049 Madrid, Spain. E-mail address: naranjo@cnb. target at DRE sites without affecting CREB-mediated tran- csic.es scription (7). DREAM, also known as potassium channel interact- The online version of this article contains supplemental material. ing protein (KChIP)-3, or calsenilin, belongs to a group of struc- Abbreviations used in this paper: BM, bone marrow; CBP, CREB-binding protein; turally and functionally related Ca2+-binding (KChIP 1 to CSR, class switch recombination; daDREAM, dominant active DREAM; DRE, downstream regulatory element; DREAM, downstream regulatory element antagonist 4) that interact with DRE sites (12), Kv4 potassium channels (13), modulator; Edem1, ER-degradation enhancing a-mannosidase-like protein; Eif4g3, and presenilins (14) regulating Ca2+-dependent transcription, chan- eukaryotic translation initiation factor 4 g, 3; ER, endoplasmic reticulum; ERAD, 2+ ER-associated degradation; GLTs, germline transcripts; HPRT, hypoxanthine-gua- nel gating, and Ca release from the ER, respectively. Because nine phosphoribosyltransferase; KChIP, potassium channel interacting protein; KO, DREAM forms heterotetramers with other KChIP proteins that knockout; N, nucleus; PI, propidium iodide; Rps9, ribosomal protein S9; UPR, un- bind DNA, daDREAM acts as a cross-dominant active mutant folded protein response. for the nuclear function of all four KChIP proteins, as previously Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 shown in neurons and T cells (10, 12). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000152 7528 DREAM REGULATES B CELL FUNCTIONS

DREAM is highly expressed in the CNS, thyroid gland, testis, and Flow cytometry analysis the thymus. Early in vitro studies described specific target genes Single-cell populations were prepared from the femurs and spleens, and Fc for DREAM repression in the brain (5) and in the thyroid gland receptors were blocked with anti-CD16/CD32. Cells (1 3 106) were stained (15). More recently, using transgenic mice expressing daDREAM in PBS/1% FCS with the respective Abs for 20 min at 4˚C. Monoclonal (hereafter TgDREAM mice), we have shown reduced proliferation Abs FITC-conjugated (B220, CD43, IgM, IgD), PE-conjugated (IgM), or and decreased IL-2, IL-4, and IFN-g production after TCR en- biotin-conjugated (CD43, IgG1, isotype of IgG1) (Pharmingen, San Diego, CA) were used. Biotin-conjugated monoclonal Abs were detected using gagement in transgenic T lymphocytes. These effects are purely streptavidin Cy-Chrome (BD Pharmingen). Analyses were performed on transcriptional and involve binding of DREAM to specific DREs an Epics XL cytometer (Coulter). located in the promoters of these cytokines (10). Qualitative and quantitative real-time RT-PCR In this study, we show that DREAM is expressed in bone marrow and mature B cells. Expression of daDREAM in B lymphocytes RNA was isolated from B cells using TRIzol (Invitrogen), treated with reduced the levels of the IgM and IgG subclasses in serum of DNAse (Ambion, Austin, TX), and reverse transcribed using hexamer primers and MMLV reverse transcriptase. To confirm the absence of ge- TgDREAM mice and increased the proliferative response in vitro. nomic DNA, each sample was processed in parallel without reverse tran- Gene expression profiling of transgenic B cells showed repression scriptase. Semiquantitative RT-PCR assays for preswitched and post- of Klf9, a cell-cycle regulator, and Eif4g3, a protein related to switched germline transcripts were performed as described (18). b-Actin translation initiation. Pulse-chase experiments suggest that a re- expression was used as a loading control. Quantitative real-time PCR for daDREAM, DREAM, KChIPs, and hypoxanthine-guanine phosphor- duction in protein synthesis rather than increased degradation ac- ibosyltransferase (HPRT) was performed as described (10). Quantification counts for the deficit in Ig production. Our results reveal a critical of Pax5, Bcl6, Xbp-1, Blimp-1, calnexin, calreticulin, BiP, CHOP, Klf9, function of DREAM- and KChIP-2–regulated genes in Ig subclass Edem1, Derlin3, Eif4g3, and Rps9 was done using specific primers and Downloaded from production and proliferation in B lymphocytes. TaqMan MGB probes (Applied Biosystems, Austin, TX). The results were normalized as indicated by parallel amplification of HPRT. Primers for quantitative RT-PCR assays preswitched and postswitched g1andε germline transcripts are reported in Supplemental Tables I and II. Speci- Materials and Methods ficity of the g1 and ε primers was determined using negative controls and by Animals the analysis of dissociation curves. Serially diluted cDNA samples were used to estimate the efficiency (Ef) of each PCR, which was 1.99 in all DREAM transgenic mice (line 1 [L1] and line 33 [L33]) were prepared by http://www.jimmunol.org/ cases. Calculation of fold differences was performed as described in Ref. 19 pronuclear microinjection in the C57BL/6xCBA hybrid background. Both Ct HPRT x 2 Ct HPRT cal and was based on the equation (1 + EfHPRT) (1 + Ef g1 transgenic lines carry a single copy of a transgenic cassette containing the ε Ct (g1orε) cal 2 Ct (g1orε) x dominant active DREAM mutant, an IRES, and the LacZ reporter gene under or -Ct) , where Ct is the number of cycles at which x the control of the CaMKIIa promoter. Transgenic lines were maintained as the threshold of fluorescence is reached, is a given sample, and cal is the heterozygotes, and age- and sex-matched littermates were used as controls. calibrator used to normalize all results (resting B cells). Expression of the transgene in both lines was found to be ubiquitous in the Western blots CNS and the immune system (10). DREAM knockout (KO) mice have been reported previously (16). The Centro Nacional de Biotecnologı´a–Consejo Purified B cells were incubated in lysis buffer (50 mM Tris-HCl, pH 7.5, Superior de Investigaciones Cientı´ficas institutional animal care and use 150 mM NaCl, 1% Nonidet P-40, protease inhibitor mixture [Roche committee approved animal protocols. Applied Science, Indianapolis, IN]) for 30 min on ice. Lysates were cleared by centrifugation. Abs against calreticulin and calnexin were from Stress- by guest on September 29, 2021 B cell isolation and stimulation gen Bioreagents, and biotin anti-mouse IgG1 (clone A85-1) was from BD Pharmingen. IgG1 was detected by streptavidin–HRP conjugate (Amer- B cells were isolated by negative selection using CD43 microbeads (MACS; sham). Cytosolic and nuclear extracts were prepared from purified B cells Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer’s using the NE-PER Extraction Reagents (Pierce, Madrid, Spain). DREAM- protocol. The purity of the different subpopulations was above 98% as specific bands were detected by Western blot using a rabbit polyclonal Ab assayed by flow cytometry. B cells were grown in RPMI with Glutamax (Ab731) prepared against aa 22–42 in the DREAM protein. This Ab rec- containing 10% FCS (Invitrogen, Barcelona, Spain), nonessential amino ognizes endogenous and mutant DREAM and does not cross-react with acids (0.1 mM), sodium pyruvate (1 mM), penicillin (100 U/ml), strepto- other members of the KChIP family. b-Actin (clone AC-15; Sigma) was mycin (100 mg/ml), and 50 mM 2-mercaptoethanol and were maintained at used as a loading control. Blots were developed by chemiluminescence 6 37˚C in an atmosphere at 5% CO2. For stimulation, B cells (0.4 3 10 ) were (ECL Advance, GE Healthcare; or SuperSignal West Dura, Pierce). cultured in 24-well plates and stimulated with LPS (20 mg/ml; Sigma, St. Louis, MO) and mouse recombinant IL-4 (10 ng/ml; BD Pharmingen, Pulse-chase experiments San Diego, CA). After 4 d in culture, IgG1 was measured by flow cytometry. B cells were stimulated with LPS plus IL-4 for 3 d and then starved for 1 h at Supernatants for IgG1 and IgE quantification were collected at day 5. For 37˚C in methionine/cysteine-free RPMI 1640 medium (Sigma) supple- CFSE labeling, resting B cells were exposed to CFSE for 30 min at 37˚C mented with 2 mM glutamine, 5% dialyzed FCS, 1 mM sodium pyruvate, (2.5 mM; Molecular Probes, Carlsbad, CA) and cultured as described. For 6 penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were labeled B cell proliferation assays, 0.2 3 10 B cells were cultured in 96-well plates, 35 3 with 1 mCi [ S]methionine/cysteine (PerkinElmer, Waltham, MA) for 60 and the cultures were pulsed for 16 h with 1 Ci [ H] (40 Ci/nmol; Amer- min at 37˚C. Cells were chased in culture medium supplemented with sham, Barcelona, Spain) before harvesting on glass fiber filters. Incorpo- excess unlabeled cysteine/methionine for 0–4 h, washed in ice-cold PBS, ration of [3H]thymidine was measured using an automatic counter (Wallac, and lysed. Supernatants were collected after centrifugation for 10 min Waltham, MA). For apoptosis analysis, B cells were stained with propidium at 13,000 rpm. IgG proteins, mostly IgG1 in B cells after LPS plus IL-4 iodide without permeabilization before analysis on an Epics XL cytometer stimulation, were captured with protein G-Sepharose (Amersham). The (Coulter, Brea, CA). For cell cycle analysis, B cells were fixed in absolute proteosome inhibitor MG132 (50 mM) was added to one sample at time 0 as ethanol precooled to 4˚C. After RNAse treatment, propidium iodide was internal control. Labeled proteins were resolved by SDS-PAGE and visu- added before analysis on an Epics XL cytometer (Coulter). For electron alized by autoradiography. microscopy analysis, B cells were stimulated with LPS plus IL-4 for 3 d and prepared as described (17). The hamster mAb against CD40 (clone HM40- RNA extraction and microarray hybridization 3) was from BD Pharmingen. RNA from naive or 2-d LPS plus IL-4 stimulated B cells was prepared using Ig ELISA TRIzol (Invitrogen) and the RNAeasy Mini Kit (Qiagen, Madrid, Spain). RNA was quantified, and the quality was assessed with a 2100 Bioanalyzer Rat anti-mouse IgG1 (LO-MG1-13), IgG2a (LO-MG2a-9), IgG2b (LO- (Agilent Technologies). cDNA was synthesized from 4 mg total RNA using MG2b-1), IgG3 (LO-MG3-13), IgM (LO-MM-3), or IgE (LO-ME-3) Abs one-cycle target labeling and control reagents (Affymetrix, Santa Clara, for coating of plates and rat anti-mouse/HRP (LO-MK-1) as secondary CA) to produce biotin-labeled cRNA. The cRNA preparation (15 mg) was detection step were from Serotec. For IgE, the secondary detection step was fragmented at 94˚C for 35 min into 35–200 bases in length. Labeled cRNAs done with biotin-conjugated rat anti-mouse IgE mAb (BD Pharmingen) and were hybridized to Affymetrix chips (GeneChip Mouse Genome 430 2.0 detected by streptavidin–HRP conjugate (Amersham). Array). Each sample was added to a hybridization solution containing 100 The Journal of Immunology 7529 mM 2-(N-morpholino) ethanesulfonic acid, 1 M Na+, and 20 mM EDTA in Results the presence of 0.01% Tween 20 to a final cRNA concentration of 0.05 mg/ Development of B lymphocytes is normal in TgDREAM mice ml. Hybridization was performed for 16 h at 45˚C. Each microarray was washed and stained with streptavidin–PE in a Fluidics station 450 (Affy- Real-time PCR analysis of whole bone marrow identified the metrix) and scanned at 1.56-mm resolution in a GeneChip Scanner 3000 7G expression of DREAM and the closely related gene KChIP-2 (Fig. System (Affymetrix). 1A). DREAM expression, however, was higher in purified B cells Microarray data analysis from the spleen compared with that of KChIP-2 (Fig. 1A). Ex- pression of KChIP-1 and KChIP-4, the other two members of the Three biological replicates were independently hybridized for each tran- scriptomic comparison. GeneChip intensities were background-corrected, KChIP family, was detected neither in bone marrow nor in purified normalized, and summarized by the robust multichip average method B cells (data not shown). Western blot analysis of cytosolic and (20) using the “Affy” package (21) from Bioconductor (http://www. nuclear fractions prepared from purified B cells showed that tet- bioconductor.org). For each comparison, moderated t test was applied rameric DREAM is localized both in the nucleus and the cytosol, to identify differentially expressed genes as implemented in the “Limma” and a monomeric DREAM band migrating approximately at 35 package (22) from Bioconductor. Raw p values were adjusted for multiple hypothesis testing using the false discovery rate method (23). Genes with kDa was observed exclusively in the cytosol (Fig. 1B). false discovery rate ,0.1 and fold changes .1.5 or ,21.5 at both 0 and 2 d To analyze the function of DREAM in B lymphocytes, we used were included in the list of induced or repressed candidates, respectively. two transgenic lines (L1 and L33) with ubiquitous expression of FIESTA (http://bioinfogp.cnb.csic.es/tools/FIESTA) viewer was used to daDREAM, in which we previously characterized a T cell phe- facilitate the application of these numerical filters and the selection of candidate genes in each comparison (24). For the biological classification notype (10). Expression level of daDREAM mRNA in purified B cells from TgDREAM mice (Fig. 1C) was 2- to 3-fold that of the of candidate genes, terms included in the “generic GO slim Downloaded from set” (S. Mundodi and A. Ireland; http://www.geneontology.org/GO.slims. endogenous DREAM mRNA levels (Fig. 1A). Total DREAM shtml) were used to group candidate genes into a reduced number of bi- protein (endogenous plus mutant DREAM) in purified transgenic ological categories. Terms from “Molecular function” name space were B cells was 1.34- and 1.50-fold higher in L1 and L33, respectively, taken into account. The Gene Ontology terms associated with the genes were obtained from the original Affymetrix annotation files available at compared with endogenous DREAM protein in wild-type B cells http://www.affymetrix.com. (Fig. 1D). Results reported hereafter correspond with experiments performed with L1 mice, which were fully comparable with data Statistical analysis from L33 mice. http://www.jimmunol.org/ Student unpaired two-tailed t test was used for statistical analyses. The To assess B cell development in TgDREAM mice, we examined p values #0.05 were considered significant. Asterisks represent statistical in detail B cell lineage populations by flow cytometry in different significance versus the appropriate control in each case: *p , 0.05; **p , organs from 8- to 12-wk-old mutant and wild-type littermates. As 0.01; ***p , 0.001. previously shown for thymus and spleen (10), the total number of cells in bone marrow was not affected by the expression of daDREAM (data not shown). In bone marrow, we analyzed B cell compartments including pro-B only (B220+CD43+), pro-B and pre- lo 2 lo lo

B (B220 IgM ) stages, as well as immature (B220 IgM ), tran- by guest on September 29, 2021 sitional (B220lo–hiIgMhi), and mature (B220hiIgMlo) B cells as described (25). B cell subsets and surface IgM labeling were similar in both transgenic and wild-type mice (Fig. 2, Supplemental Fig. 1). Likewise, we did not observe differences in the mature B cell compartment (IgM+ IgD+) in the spleen or when we dis- tinguished follicular B cells (B220+CD23+CD21lo) from marginal zone B cells (B220+CD23lo/2CD21hi) (Fig. 2, Supplemental Fig. 1). Evaluation of the B1 cell compartment (B220+CD5+IgM+)in the peritoneal cavity also showed comparable percentages and cell numbers in transgenic and wild-type mice (data not shown). Thus, similar to the effect reported for the T cell lineage (10), we con- clude that expression of daDREAM does not affect B lymphocyte development in vivo. Serum Ig levels are reduced in TgDREAM mice We next studied whether daDREAM expression interferes with FIGURE 1. DREAM is expressed in B lymphocytes. A, Quantitative B cell Ig secretory function. We measured steady-state levels of real-time PCR analysis of DREAM and KChIP-2 mRNA levels in bone circulating Ig isotypes in TgDREAM mice. Serum levels of IgG marrow (BM) and purified B cells from wild-type mice. Results are the subclasses (1, 2a, 2b, and 3) and IgM were significantly decreased in mean 6 SD of four mice. B, Western blot analysis of cytosolic and nuclear transgenic compared with wild-type mice (Fig. 3). The inhibition extracts (nonreducing PAGE) from purified B cells prepared from wild- accounted for 53, 20, 55, 68, and 64%, respectively, in 8- to 12-wk- type mice. As control, B cell extracts from DREAM KO mice were pro- old mice and was maintained throughout the life span of the mice, cessed in parallel. Arrowheads mark the migration of DREAM-specific with similarly reduced levels in 1-y-old mice (data not shown). We immunoreactive bands: tetramer DREAM (solid) and monomer DREAM (open). Nonspecific bands are marked by asterisk. b-Actin is shown as conclude that expression of daDREAM reduces the levels of IgG loading control. C, Quantitative real-time PCR analysis of daDREAM subclasses and IgM in vivo. expression in purified B transgenic cells from mouse lines L1 and L33. Decreased Ig production in TgDREAM mice is due to an Results are the mean 6 SD of four mice. D, Western blot analysis (re- intrinsic B cell defect ducing PAGE) of DREAM in total extracts from purified B cells from wild-type, L1, and L33. The DREAM Ab recognizes endogenous and Because T cell function is impaired in TgDREAM mice (10), we mutant DREAM. Levels of b-actin were analyzed for normalization, and investigated if the reduced Ig levels in TgDREAM serum are in- the mean ratios of DREAM/b-actin of four mice per genotype are shown. trinsic to B cells. For this, we assayed in vitro purified B cells for 7530 DREAM REGULATES B CELL FUNCTIONS

FIGURE 2. B cell development in bone marrow and spleen in TgDREAM mice is normal. Bone marrow and spleen were obtained from 8- to 12- wk-old mice, and single-cell suspensions were stained for flow cytometry with the indicated Ab combinations. Positive cells (%) within a quadrant are indicated. The results are representative of four wild-type and four TgDREAM mice. Downloaded from their ability to secrete Ig upon appropriate activation. For these pared with that of wild type (Fig. 4D), confirming that impaired experiments, we stimulated splenic purified B cells derived from ability to produce IgG1 in vitro is due to an intrinsic defect of TgDREAM and wild-type mice with LPS alone and with LPS to- transgenic B cells. gether with IL-4 or IFN-g and quantified secreted Ig isotypes in the In vitro proliferation of TgDREAM B lymphocytes is increased supernatant after 5 d. Stimulation with LPS alone induces class switch recombination (CSR) and production of IgG2b and IgG3 Because Ig levels were reduced, we next analyzed the proliferative http://www.jimmunol.org/ isotypes in wild-type B cells, whereas activation with LPS together response of transgenic B cells to mitogenic stimuli. Surprisingly, with IL-4 or IFN-g induces CSR and production of respectively transgenic splenocytes proliferated faster than wild-type cells in IgG1/IgE and IgG2a. We observed that Ig secretion was markedly response to BCR stimulation (anti-IgM), to anti-CD40 or to LPS reduced in culture supernatants from LPS plus IL-4 stimulated without or with IL-4, as assessed by thymidine incorporation (Fig. transgenic B cells (Fig. 4A) and LPS plus IFN-g (Supplemental 5A). This increased proliferative response was observed also in Fig. 2). After LPS stimulation, however, this defect was less pro- purified transgenic B cells compared with that of wild type after nounced and only significant for IgM (Supplemental Fig. 2). LPS without or with IL-4 stimulation (Fig. 5B). To assess the ba- To dissect the mechanism responsible for the Ig production sis for this phenotype, we examined the effect of daDREAM on defect, we focused on IgG production obtained after LPS plus IL-4 B cell division. CFSE analysis revealed that cell division of trans- by guest on September 29, 2021 stimulation. We measured the proportion of cells expressing sur- genic B cells after LPS plus IL-4 stimulation was accelerated face IgG1 after 4 d of stimulation and found a significant (p , compared with that of wild-type B cells. Two days after stimu- 0.001) decrease in IgG1+ cells in transgenic cultures (5.8 6 0.4%) lation, the percentages of dividing cells were 56 and 27% for compared with that of wild type (9.3 6 0.2%) (Fig. 4B). Of note, TgDREAM and wild-type B cells, respectively (Fig. 5C). the intensity (mean fluorescence intensity) of the IgG1 labeling is To investigate whether the increased proliferation is accompa- similar in wild-type and transgenic B IgG1+ cells, suggesting that nied with accelerated death, we quantified the number of apoptotic it is not the amount of membrane IgG1 per cell but the frequency cells by propidium iodide staining. We found that the number of of IgG1+ cells that is affected in transgenic B cells. We analyzed apoptotic cells was similar in transgenic and wild-type B cells at all the number of IgG1+ cells in each cell division after LPS plus IL-4 time points analyzed after stimulation (Fig. 5D,5E), suggesting stimulation and found that the percentage of IgG1+ transgenic that apoptosis is not increased in transgenic B cells. These results B cells was significantly decreased after four divisions (Fig. 4C). indicate that daDREAM leads to an acceleration of B cell division The total content of intracellular IgG1 proteins was analyzed by associated with increased proliferation but does not result in in- Western blot using b-actin protein as a loading control. The IgG1 creased susceptibility to cell death. protein level was decreased by 35% in transgenic B cells com- Functional redundancy between DREAM and KChIP-2 in B cells Because B cells express both DREAM and KChIP-2 genes, we searched for a functional phenotype in B cells from DREAM- deficient mice, which could suggest specific roles for these two KChIP family members in B cells. Analysis of Ig levels in serum showed no difference between DREAM2/2 and wild-type mice (Fig. 6A). Likewise, secretion of IgG1 from purified B cells into culture medium was not different in KO and wild-type B cell cultures (Fig. 6B), and CFSE analysis did not reveal significant differences in the percentages of dividing cells between KO and wild-type B cells (Fig. 6C). Thus, Ig production and B cell pro- FIGURE 3. Decreased serum Ig levels in TgDREAM mice. Classes of 2/2 Ig were quantified in serum from six wild-type (open symbols) and six liferation were normal in DREAM mice. These data suggest TgDREAM (solid symbols) mice. Concentration of the different Ig values a functional redundancy between DREAM and KChIP-2 in the is represented on logarithmic scale. Statistical significance versus corre- control of Ig production and proliferation in B lymphocytes, as sponding control: *p , 0.05; **p , 0.01; ***p , 0.001. previously shown in T cells (10). The Journal of Immunology 7531 Downloaded from http://www.jimmunol.org/

FIGURE 4. Impaired IgG1 and IgE secretion is due to an intrinsic defect in transgenic B cells. A, B cells were stimulated with LPS plus IL-4 for 5 d, FIGURE 5. Increased proliferative response in transgenic B cells in vitro. and Ig concentration in culture supernatants was assessed by ELISA. B, A, Proliferation of splenocytes 2 d after stimulation with IgM, CD40, or LPS

LPS plus IL-4 stimulated B cells from 4-d cultures were stained for IgG1 without or with IL-4, assessed by [3H]thymidine incorporation. B, Pro- by guest on September 29, 2021 and analyzed by flow cytometry. The graph represents the proportion of liferation of purified B cells stimulated for 2 d by LPS plus IL-4, assessed by + IgG1 cells; results are the mean 6 SD of six mice in two separate ex- [3H]thymidine incorporation. C, CFSE-labeled purified B cells were stimu- periments. A representative histogram is shown. C, The proportion of lated for 2 d with LPS plus IL-4. Histograms showing the CFSE profile are + IgG1 cells in each division cycle from the dot plot of CFSE versus surface representative of four wild-type and four TgDREAM mice. Graphs show the labeling with IgG1. Results are the mean 6 SD of four mice and are proportion of cells present in each cell division. D, Apoptosis analysis by representative of two experiments. D, Western blot analysis of IgG1 in propidium iodide (PI) staining of B cells at different days after stimulation whole-cell extracts from wild-type and transgenic B cells treated with LPS with LPS plus IL-4. Results are the mean 6 SD of four mice and are rep- plus IL-4 during 3 d. Purified IgG1 was included as a control, and b-actin resentative of two experiments. E, Quantification by PI incorporation of levels were used as loading control. The ratio IgG1/b-actin is shown below B cells in G0-1, S, and G2-M phases of the cell cycle 3 d after stimulation each lane. The average decrease (35%) in IgG1 content is the result from with LPS plus IL-4. Results are the mean 6 SD of four mice and are rep- six mice in two separate experiments. Statistical significance versus cor- resentative of two experiments. Statistical significance versus corresponding responding control (A–C): ***p , 0.001. control (A–C, E): *p , 0.05; **p , 0.01; ***p , 0.001.

Transgenic B cells do not exhibit defects in IgH locus transcription TgDREAM B splenocytes undergo normal plasma cell Because it has been shown that daDREAM functions as a tran- differentiation scriptional repressor in T lymphocytes (10), we investigated if the To verify if the transgenic B cell hyperproliferative response invitro IgH locus is a direct target of DREAM in B cells. For this purpose, could influence the differentiation into secretory plasma cells, we we performed an extended analysis of the multiple IgH transcripts quantified the expression levels of genes known to be critical for the induced after LPS plus IL-4 stimulation. differentiation toward plasmocytes. Xbp-1 and Blimp-1 are key We monitored the various germline transcripts (GLTs) initiated regulators promoting plasmocytic development, whereas Pax5 and from the I promoters upstream of donor and acceptor switch regions Bcl6 inhibit plasma cell differentiation (3). We found that down- and associated with CSR. The preswitch GLT (Im-Cm,Ig1-Cg1, regulation of Pax5 and Bcl6 and increased expression of Xbp-1 and Iε-Cε) and the CSR-resulting postswitch hybrid transcripts and Blimp-1 occurred to the same extent in both wild-type and (Im-Cg1 and Im-Cε) were quantified at days 3 and 4, respectively. transgenic B cells stimulated with LPS plus IL-4 at days 3 and Semiquantitative PCR not only showed that both preswitch GLT 5(Fig.7A). In addition, the percentage of cells that express and postswitch HT were not decreased, but also that Iε-Cε pre- syndecan-1 (CD138), a marker of Ab-secreting cells, was identical switch germline transcription was increased in transgenic B cells in stimulated TgDREAM and wild-type B cells (Fig. 7B). These compared with that in wild-type B cells (Fig. 8A,8B). These data data indicate that TgDREAM B cell cultures have similar ability as support the notion that the reduced Ig production after LPS plus wild-type B cells to differentiate into plasma cells in vitro. IL-4 stimulation is not due to defective CSR. Using real-time 7532 DREAM REGULATES B CELL FUNCTIONS Downloaded from

FIGURE 6. Lack of a B cell phenotype in DREAM2/2 mice. A,Ig levels were quantified in serum from six wild-type (open symbols) and six DREAM2/2 (gray symbols) mice. Concentration of the different Ig values is represented on logarithmic scale. B, Isolated DREAM2/2 B cells were stimulated with LPS plus IL-4 for 5 d, and Ig concentration in culture http://www.jimmunol.org/ supernatants was assessed by ELISA. Results are the mean 6 SD of four mice. C, CFSE-labeled purified B cells were stimulated with LPS plus IL- FIGURE 7. Normal plasma cell differentiation of transgenic B cells. A, 4. Graphs show the proportion of cells present in each cell division and are Quantitative real-time PCR analysis of Pax5, Bcl6, Xbp-1, and Blimp-1 the mean 6 SD of four mice. mRNA expression in B cells stimulated by LPS plus IL-4 during the in- dicated times. B, Flow cytometry analysis of syndecan-1 (CD138)-positive B cells after 4 d of stimulation with LPS plus IL-4. Results are repre- PCR, we confirmed that Iε-Cε preswitch germline is increased sentative of four wild-type and four TgDREAM mice. Graph shows the (data not shown) and found that Ig1-Cg1 (preswitch GLT) also proportion of syndecan-1 intermediate (I) and high (H) cells. Results are was higher in transgenic than in wild-type B cells (Fig. 8C), de- the mean 6 SD of four mice and are representative of three experiments. spite the reduced number of IgG1-expressing cells and IgG1 by guest on September 29, 2021 protein secretion (Fig. 4B,4D). To investigate whether the stability of Ig proteins is affected in To analyze total H chain transcription in B cells after in vitro transgenic B cells, we performed pulse-chase experiments after stimulation, we measured RNA products transcribed from any 3 d of stimulation with LPS plus IL-4. We observed that incor- functionally rearranged VDJ segment (corresponding with the ex- poration of labeled amino acids was reduced in transgenic B cells, pressed allele) by real-time PCR. Amplification was performed and accumulation of labeled IgG1 protein at time 0 was repro- using a combination of forward primers located in each JH seg- ducibly decreased (26.7 6 0.4%) in transgenic compared with ment (JH1 to JH4) and reverse primers located in g1 and ε constant wild-type B cells (Fig. 10A,10B). Importantly, decay of labeled genes. At days 4 and 5 of stimulation, IgH transcripts g1 and ε IgG1 protein both at 2 and 4 h after the pulse was not different in were not decreased in transgenic B cell cultures; on the contrary, transgenic and wild-type B cells compared with respective values Ig g1 was increased in transgenic B cells (Fig. 8D). Taken to- at time 0 (Fig. 10A,10B). Inhibition of the proteosome activity gether, these results indicate that IgH transcription is not impaired with MG132 blocked protein decay to a similar extent in both in transgenic B cells after in vitro stimulation, suggesting that transgenic and wild-type B cells (Fig. 10A,10B). Taken together, daDREAM does not repress IgH locus promoters. these results indicate that reduced Ig production is not related to impaired activation of the secretory pathway or increased degra- Ig secretion and degradation processes are normal in dation process. stimulated transgenic B cells Given that IgG1/IgE secretion is decreased in TgDREAM B cell Transcriptomic analysis in transgenic B cells cultures without decrease in the corresponding mRNA levels, we Because daDREAM has been described as a dominant active mutant examined if the ER expansion necessary for Ig secretion is af- for the transcriptional repressor function of DREAM/KChIP-2 (10), fected in transgenic B cells. Electron microscopy analysis, how- we searched for potential transcriptional targets that could help to ever, showed that the increase in cell size, largely due to cytosolic understand the observed phenotype in transgenic B cells. Using expansion of the ER, 3 d after stimulation with LPS plus IL-4 was cDNA microarrays, we compared global gene expression in wild- similar in wild-type and transgenic B cells (Fig. 9A). typeandtransgenicBcells,freshlyisolatedand2dafterLPSplus ER enlargement during plasma cell differentiation involves IL-4 stimulation. The complete set of genes whose expression levels the preferential expression of specific proteins, including ER chap- were altered in transgenic B cells is stored in the Gene Expression erones, BiP, calnexin, and calreticulin and the transcription factors Omnibus database (accession no. GSE16874; http://www.ncbi.nlm. Xbp-1 and CHOP, which participate in the UPR. Confirming the nih.gov/geo/query/acc.cgi?acc=GSE16874). Upregulated and down- electron microscopy data, real-time PCR and Western blot analysis regulated genes both at days 0 and 2 were categorized according to showed that expression of these UPR-related genes was similar in Gene Ontology terms and are presented in Supplemental Table III transgenic and wild-type B cells (Fig. 9B,9C). and Fig. 11A and 11B. The Journal of Immunology 7533 Downloaded from

FIGURE 8. CSR and Ig gene transcription efficiency is not reduced in http://www.jimmunol.org/ transgenic B cells. A, Semiquantitative RT-PCR for Im-Cm,Ig1-Cg1, and Iε-Cε preswitch GLTs in wild-type and transgenic B cells stimulated with LPS plus IL-4 for 4 d. B, Semiquantitative RT-PCR for postswitch Im-Cg1 hybrid transcripts in wild-type and transgenic B cells stimulated with LPS plus IL-4 for 3 d. Results from the scanning of the gels after semi- quantitative RT-PCR are shown to the left. These results are from six mice in two separate experiments and are corrected by b-actin expression, used as loading control. C, Real-time PCR for Ig1-Cg1 preswitch GLTs in wild- type and transgenic B cells stimulated with LPS plus IL-4 for 3 d. Results are expressed as fold induction relative to resting B cells. Results are the by guest on September 29, 2021 6 mean SD of four mice and are representative of three experiments. D, FIGURE 9. ER expansion and chaperone protein expression is not af- Real-time PCR for g1 H chain transcript in wild-type and transgenic B fected in stimulated transgenic B cells. A, Electron microscopy analysis of cells stimulated with LPS plus IL-4 for 4 d. Results are expressed as fold ER enlargement in unstimulated and LPS plus IL-4 stimulated wild-type 6 induction relative to resting B cells. Results are the mean SD of four and transgenic B cells. Scale bars, 0.5 mm. B, Quantitative real-time PCR mice and are representative of three experiments. Statistical significance of calnexin, calreticulin, BiP, and CHOP transcripts in wild-type and , , , versus corresponding control (C): *p 0.05; **p 0.01; ***p 0.001. transgenic B cells stimulated with LPS plus IL-4 for 3 d. Results are the mean 6 SD of four mice and are representative of three experiments. C, Western blot analysis with anti-calnexin and anti-calreticulin Abs in We first searched for cell cycle-related genes that could explain B cells isolated from three wild-type and three transgenic mice and stim- the increased proliferation observed in transgenic B cells. In the ulated with LPS plus IL-4 during 3 d. Expression levels of actin were used cluster of transcription-related genes, which accounts for 24% as a control. Results are representative of three experiments. N, nucleus. of downregulated genes, we observed downregulation of Klf9, a cell-cycle regulatory gene, in both unstimulated and stimulated transgenic B cells (Supplemental Table III). Reduced expression of synthesis, we investigated changes in the expression of eukaryotic Klf9 in quiescent memory human B cells has been associated with translation initiation factor 4 g, 3 (Eif4g3), an integral scaffold earlier entry in cell division and faster proliferation (26). By real- protein of the translation initiation machinery, and ribosomal pro- time PCR, we confirmed that Klf9 expression is reduced in trans- tein S9 (Rps9) by real-time PCR. Notably, a significant reduction in genic B cells compared with that in wild-type, both in unstimulated Eif4g3 mRNA levels was found in transgenic unstimulated B cells and stimulated conditions (Fig. 12A). Furthermore, Klf9 expression (Fig. 12B). However, 3 d after LPS plus IL-4 stimulation, levels of was decreased also during wild-type B cell differentiation (Fig. this transcript were reduced in both genotypes, and no difference 12A). These results indicate that Kfl9 repression is associated with was found between wild-type and transgenic cells (Fig. 12B). By B cell stimulation and that reduced Klf9 levels in unstimulated contrast, expression of Rps9 was similar in both genotypes before transgenic B cells could be responsible for the accelerated entry and after stimulation (Supplemental Fig. 3). These data suggest that in cell division and increased proliferation. some genes implicated in protein translation (e.g., Eif4g3) could be Among other genes with modified expression, we focused our directly or indirectly targeted by DREAM in B cells. Concerning attention on genes and pathways that could be involved in the de- genes related to protein degradation, microarray data showed that crease in Ig protein levels in transgenic B cells. Transcriptomic expression of ER-degradation enhancing a-mannosidase-like pro- analysis suggested that several genes involved in protein synthesis tein (Edem1) and Derlin3 were increased in transgenic B cells (Fig. (Fig. 11A, Supplemental Table III) and protein degradation (Fig. 11B). These genes encode key components of the ER-associated 11B, Supplemental Table III) were modified in transgenic com- degradation (ERAD) pathway, and it has been shown that enhanced pared with wild-type B cells. Concerning genes related to protein levels of Edem1 (27, 28) and Derlin3 (29) are associated with 7534 DREAM REGULATES B CELL FUNCTIONS

DREAM in basal conditions is a regulated process, which varies in different cell types (30). In this study, we show that in unstimu- lated B cells, DREAM is located in the nucleus exclusively as a tetramer, its preferred DNA-binding configuration (31), whereas in the cytosol, tetramer and monomer DREAM coexist. Notably, monomeric DREAM in the cytosol from B cells migrates slower than theoretically expected, suggesting posttranslational modifi- cations, which may be related to its exclusion from the nucleus. Previous studies have shown that expression of daDREAM has a cross-dominant effect and blocks the function of endogenous KChIP proteins when present in a ratio as low as 1/3 (10). In this study, we show that in transgenic B cells, the daDREAM/DREAM protein ratio is 1/3 and 1/2 in L1 and L33, respectively. Because KChIP-2 expression in wild-type B cells is lower than that of DREAM, the expression of the dominant active mutant in the transgenic B cells should be sufficient to block endogenous FIGURE 10. Translation of IgG1 in transgenic B cells is reduced. A, DREAM/KChIP-3 and KChIP-2 function. Furthermore, this - Pulse-chase analysis of Ig levels in wild-type and transgenic B cells after stimulation with LPS plus IL-4 for 3 d. A representative autoradiogram tively low expression level may prevent unwanted effects related from one of three experiments is shown. Ig H and L chains are indicated. to transgene overexpression. Downloaded from Levels of Igs were analyzed at 0, 2, and 4 h after the pulse with labeled Gene profiling analysis comparing wild-type and transgenic amino acids. A control sample at 4 h after the pulse in the presence of the B cells showed specific changes in gene expression in the trans- proteosome inhibitor MG132 (lane 4i) is also shown. B, Quantitative genic population, both unstimulated and 2 d after stimulation. analysis of pulse-chase experiments. Bands corresponding with the H Activation of gene expression in transgenic B cells might be due chain from three separate pulse-chase experiments were scanned, and to de-repression mechanisms related to the inability of daDREAM absolute OD values are shown. Statistical significance: **p , 0.01. to block CREB constitutively (7), or to other transcriptional activa- http://www.jimmunol.org/ tors, or to exclusion from repressor complexes yet to be character- ized. Gene repression, however, can be related to the direct domi- accelerated degradation of misfolded glycoproteins. Use of real- nant active effect at DRE sites by the Ca2+- and cAMP-insensitive time PCR confirmed the induction of both Edem1 and Derlin3 in “constitutive” repressor daDREAM. Of note, the largest percentage transgenic B cells (Fig. 12C,12D). Thus, microarray data disclosed of repressed genes (24%) corresponds with transcription factors different mechanisms to explain defective Ig production in trans- suggesting i) a role for DREAM as a master-repressor switch in genic B cells. B cells and ii) that some of the changes in gene expression corre- spond with genes that are not direct DREAM targets. Whether Discussion these specific effects on gene expression revealed by the use of by guest on September 29, 2021 DREAM is a Ca2+-binding protein with distinct functions in mutated DREAM take place in heterochromatin as well as in eu- different cellular compartments. The subcellular localization of chromatin domains remains to be further analyzed.

FIGURE 11. Transcriptomic analysis in transgenic B cells. A, Functional clustering (Gene Ontology) of genes with modified expression encoding proteins related to protein synthesis. Biological triplicates at days 0 and 2 are presented as a “heat map.” B, Functional clustering (Gene Ontology) of upregulated genes encoding proteins located in the ER. Biological triplicates at days 0 and 2 are presented as a “heat map.” The Journal of Immunology 7535

a role in the reduction of T-dependent Ig levels in the serum in vivo. Because Ig transcription is not altered in transgenic B cells, the reduced Ig secretion observed points toward a defect in the Ig protein synthesis. Gene profiling identified decreased expression of Eif4g3 and disclosed a potential mechanism for decreased Ig protein synthesis in primary splenic B cells from TgDREAM mice. Eif4g are scaffold proteins of the translation initiation machinery that connect mRNA and the 40S ribosomal subunit to initiate translation. The molecular basis for a potentially specific effect of reduced Eif4g3 expression on Ig production without a general effect on protein synthesis is currently unknown. It has recently been reported (34), however, that a single amino acid mutation in the W2 domain of Eif4g3 selectively blocks the translation of Hspa2, a chaperone of CDC2A kinase that together with cyclin B regulates the G2/M transition and the exit from meiotic prophase FIGURE 12. Changes in gene expression in transgenic B cells. Quan- (35). Lack of Hspa2 protein abrogates CDC2A activity, which titative real-time PCR for (A) Klf9, (B) Eif4g3, (C) Edem1, and (D) results in meiotic arrest of mouse spermatocytes and male in-

Derlin3 mRNA in wild-type and transgenic B cells stimulated with LPS fertility (34). How mutant Eif4g3 specifically affects translation of Downloaded from 6 plus IL-4 for the indicated times. Results are the mean SD of four mice specific mRNAs is not resolved, though signals contained in the and are representative of two experiments. Statistical significance versus long 59 leader sequence of the Hspa2 transcript are probably re- corresponding control (A–D): *p , 0.05; **p , 0.01; ***p , 0.001. sponsible. Whether this is the case for Ig transcripts is not resolved either. Lack of a specific Ab for Eif4g3 precluded the analysis of DREAM, also named KChIP-3, is a member of the DREAM/ Eif4g3 protein levels to support further its role in Ig translation in 2+ KChIP family of neuronal Ca sensors, KChIP 1 to 4 (13). All basal conditions and after B cell stimulation. http://www.jimmunol.org/ four DREAM/KChIP genes encode structurally related proteins Gene profiling also identified the upregulation in transgenic able to bind DRE sites and block DRE-dependent transcription B cells of Edem1 and Derlin3, two key components of the ERAD (12). In the immune system, DREAM and KChIP-2 are the only pathway. Edem1 recognizes the Man8GlcNAc2 structure, present family members expressed. The functional redundancy between in misfolded glycoproteins, and targets them to the proteasome DREAM and KChIP-2 and their coexpression in lymphocytes may (27, 36, 37), and Derlin3, an ER membrane-spanning protein, fa- account for the lack of phenotype reported in the immune system cilitates the cytosolic translocation of ERAD substrates (29). Over- of DREAM KO mice (16). In the current study, DREAM-deficient expression of either Edem1 (36) or Derlin3 (29) accelerates, and mice did not show modification in the levels of serum Igs or in Ig small interfering RNA for Derlin3 (29) reduces, the degradation of secretion after B cell stimulation. In TgDREAM mice, however, misfolded glycoproteins. Although pulse-chase experiments did by guest on September 29, 2021 expression of the dominant active mutant blocks repressor activity not support increased degradation of IgG as the main mechanism of both endogenous DREAM and KChIP-2 proteins in the nucleus to explain reduced Ig levels in transgenic B cells in vitro, we cannot resulting in an apparent phenotype. Nevertheless, the functional exclude that increased levels of Edem1 or Derlin3 observed in identity among KChIP family members is not absolute, and recent transgenic B cells could participate in the Ig production defect in studies have shown differences in their subcellular localization vivo. The biological significance of Edem1 and Derlin3 upregu- and in their functions outside the nucleus (e.g., Ca2+-dependent lation in DREAM transgenic B cells is currently not understood. exocytosis in PC12 cells) (32). Thus, subtle functional differences Stimulated transgenic B cells show an accelerated cell division between DREAM and KChIP-2 in B cell gene regulation cannot compared with that of wild type and increased proliferative re- be entirely excluded by the absence of a phenotype in DREAM- sponse without change in apoptosis susceptibility. Microarray deficient B cells. analysis identified the downregulation of Klf9 in unstimulated TgDREAM mice showed a global decrease in serum Ig levels. and stimulated transgenic B cells. Klf9 is important to maintain We believe that this phenotype is physiologically relevant because cell quiescence and to control cell division in human B cells (26). preliminary results from asthma models show that the production Furthermore, we found that expression of Klf9 was decreased of Ag-specific IgG1 is reduced in TgDREAM mice (J.R. Naranjo, also in wild-type B cells after stimulation, suggesting that the unpublished data) suggesting that inhibition of DREAM activity in downregulation of Klf9 is a prerequisite for mouse B cell pro- B cells may have therapeutic potential. In vitro experiments using liferation. The proximal promoter of the Klf9 gene contains DRE purified B cells stimulated with LPS 6 IL-4 showed that the defect elements that could participate in the transcriptional repression in Ig production is intrinsic to B cells. Deregulation of the Ig by daDREAM. Ectopic expression of Klf9 in human B cells production in transgenic B cells appears not to be related to reduces the number of B cells beingrecruitedintodivisionand changes in the plasma cell differentiation program, as expression delays their entry into division (26). Together, these data suggest of Blimp-1 and Xbp-1, two proteins heavily implicated in plasma that Klf9 could maintain cellular quiescence in naive B cells and cell differentiation and Ig secretion, is normal in transgenic that its downregulation allows transgenic B cells to enter di- B cells. Furthermore, deregulation of Ig production in transgenic vision earlier than wild type. B cells is not due to a transcription defect because multiple tran- Ca2+-dependent changes in gene expression are essential for scripts arising from the IgH locus (germline and VDJ-rearranged the immune response. Two major transcription factors, NFAT and H chain transcripts) were increased in transgenic B cells. In- , have in the past been considered to carry out these changes creased GLT and IgH mRNA levels in transgenic B cells could be (38–40). Our results in B cells and a previous study focused on related to accelerated cell division (33). Because previous work T lymphocytes (10) indicate that DREAM also participates in the with the TgDREAM mice showed a defect in cytokine production Ca2+-dependent control of the immune response and is a potential and T cell proliferation (10), we cannot exclude that T cells play new target for immunosuppressant drugs. Further studies should 7536 DREAM REGULATES B CELL FUNCTIONS analyze the functional relations between DREAM, NFAT, and triggered in human cells by HCV polyprotein expression from a viral vector. 2+ Virol. J. 5: 102. MEF2, which might direct a global Ca -dependent transcriptional 18. Pinaud, E., A. A. Khamlichi, C. Le Morvan, M. Drouet, V. Nalesso, M. Le Bert, network controlling the immune response. and M. Cogne´. 2001. Localization of the 39 IgH locus elements that effect long- distance regulation of class switch recombination. Immunity 15: 187–199. 19. Reina-San-Martin, B., S. Difilippantonio, L. Hanitsch, R. F. Masilamani, Acknowledgments A. Nussenzweig, and M. C. Nussenzweig. 2003. H2AX is required for re- We thank C. Patin˜o for electron microscopy experiments, Drs. L. Pelletier and combination between immunoglobulin switch regions but not for intra-switch P. Druet for critical reading of the manuscript, and R. Susı´nandP.Gonza´lez region recombination or somatic hypermutation. J. Exp. Med. 197: 1767– 1778. for technical assistance. 20. Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U. Scherf, and T. P. Speed. 2003. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249–264. Disclosures 21. Gautier, L., L. Cope, B. M. Bolstad, and R. A. Irizarry. 2004. affy—analysis of The authors have no financial conflicts of interest. Affymetrix GeneChip data at the probe level. Bioinformatics 20: 307–315. 22. Smyth, G. K. 2005. Limma: Linear Models for Microarray Data. Springer, New York. References 23. Benjamini, Y., and Y. Hochberg. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B 1. Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune Stat. Methodol. 57: 289–300. response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of 24. Oliveros, J. C. 2007. FIESTA@BioinfoGP. An interactive server for analyzing responding cell populations. J. Exp. Med. 173: 1165–1175. DNA microarray experiments with replicates. Available at: http://bioinfogp.cnb. 2. Smith, K. G., A. Light, G. J. Nossal, and D. M. Tarlinton. 1997. The extent of csic.es/tools/FIESTA. Accessed October 16, 2010. affinity maturation differs between the memory and antibody-forming cell 25. Hoffmann, A., S. Kerr, J. Jellusova, J. Zhang, F. Weisel, U. Wellmann, compartments in the primary immune response. EMBO J. 16: 2996–3006. T. H. Winkler, B. Kneitz, P. R. Crocker, and L. Nitschke. 2007. Siglec-G is Downloaded from 3. Tarlinton, D., A. Radbruch, F. Hiepe, and T. Do¨rner. 2008. Plasma cell differ- a B1 cell-inhibitory that controls expansion and calcium signaling of the entiation and survival. Curr. Opin. Immunol. 20: 162–169. B1 cell population. Nat. Immunol. 8: 695–704. 4. Brewer, J. W., and L. M. Hendershot. 2005. Building an antibody factory: a job 26. Good, K. L., and S. G. Tangye. 2007. Decreased expression of Kruppel-like for the unfolded protein response. Nat. Immunol. 6: 23–29. factors in memory B cells induces the rapid response typical of secondary an- 5. Carrio´n, A. M., W. A. Link, F. Ledo, B. Mellstro¨m, and J. R. Naranjo. 1999. tibody responses. Proc. Natl. Acad. Sci. USA 104: 13420–13425. DREAM is a Ca2+-regulated transcriptional repressor. Nature 398: 80–84. 27. Molinari, M., V. Calanca, C. Galli, P. Lucca, and P. Paganetti. 2003. Role of 6. Ledo, F., A. M. Carrio´n, W. A. Link, B. Mellstro¨m, and J. R. Naranjo. 2000. EDEM in the release of misfolded glycoproteins from the calnexin cycle. Sci- DREAM-alphaCREM interaction via leucine-charged domains derepresses ence 299: 1397–1400. downstream regulatory element-dependent transcription. Mol. Cell. Biol. 20: 28. Olivari, S., T. Cali, K. E. Salo, P. Paganetti, L. W. Ruddock, and M. Molinari. 2006. http://www.jimmunol.org/ 9120–9126. EDEM1 regulates ER-associated degradation by accelerating de-mannosylation 7. Ledo, F., L. Kremer, B. Mellstro¨m, and J. R. Naranjo. 2002. Ca2+-dependent of folding-defective polypeptides and by inhibiting their covalent aggregation. Bio- block of CREB-CBP transcription by repressor DREAM. EMBO J. 21: 4583– chem.Biophys.Res.Commun.349: 1278–1284. 4592. 29. Oda, Y., T. Okada, H. Yoshida, R. J. Kaufman, K. Nagata, and K. Mori. 2006. 8. Sanz, C., B. Mellstrom, W. A. Link, J. R. Naranjo, and J. L. Fernandez-Luna. Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response 2001. Interleukin 3-dependent activation of DREAM is involved in transcrip- and are required for ER-associated degradation. J. Cell Biol. 172: 383–393. tional silencing of the apoptotic Hrk gene in hematopoietic progenitor cells. 30. Zaidi, N. F., E. E. Thomson, E. K. Choi, J. D. Buxbaum, and W. Wasco. 2004. EMBO J. 20: 2286–2292. Intracellular calcium modulates the nuclear translocation of calsenilin. J. Neu- 9. Ledo, F., W. A. Link, A. M. Carrio´n, V. Echeverria, B. Mellstro¨m, and rochem. 89: 593–601. J. R. Naranjo. 2000. The DREAM-DRE interaction: key nucleotides and dom- 31. Osawa, M., K. I. Tong, C. Lilliehook, W. Wasco, J. D. Buxbaum, H. Y. Cheng, inant negative mutants. Biochim. Biophys. Acta 1498: 162–168. J. M. Penninger, M. Ikura, and J. B. Ames. 2001. Calcium-regulated DNA 10. Savignac, M., B. Pintado, A. Gutierrez-Adan, M. Palczewska, B. Mellstro¨m, and binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/ by guest on September 29, 2021 J. R. Naranjo. 2005. Transcriptional repressor DREAM regulates T-lymphocyte DREAM/KChIP3. J. Biol. Chem. 276: 41005–41013. proliferation and cytokine gene expression. EMBO J. 24: 3555–3564. 32. Venn, N., L. P. Haynes, and R. D. Burgoyne. 2008. Specific effects of KChIP3/ 11. Gomez-Villafuertes, R., B. Torres, J. Barrio, M. Savignac, N. Gabellini, F. Rizzato, calsenilin/DREAM, but not KChIPs 1, 2 and 4, on calcium signalling and reg- B. Pintado, A. Gutierrez-Adan, B. Mellstro¨m, E. Carafoli, and J. R. Naranjo. 2005. ulated secretion in PC12 cells. Biochem. J. 413: 71–80. Downstream regulatory element antagonist modulator regulates Ca2+ homeostasis 33. Hodgkin, P. D., J. H. Lee, and A. B. Lyons. 1996. B cell differentiation and and viability in cerebellar neurons. J. Neurosci. 25: 10822–10830. isotype switching is related to division cycle number. J. Exp. Med. 184: 277–281. 12. Link, W. A., F. Ledo, B. Torres, M. Palczewska, T. M. Madsen, M. Savignac, 34. Sun, F., K. Palmer, and M. A. Handel. 2010. Mutation of Eif4g3, encoding J. P. Albar, B. Mellstro¨m, and J. R. Naranjo. 2004. Day-night changes in a eukaryotic translation initiation factor, causes male infertility and meiotic ar- downstream regulatory element antagonist modulator/potassium channel inter- rest of mouse spermatocytes. Development 137: 1699–1707. acting protein activity contribute to circadian gene expression in pineal gland. J. 35. Sun, F., and M. A. Handel. 2008. Regulation of the meiotic prophase I to Neurosci. 24: 5346–5355. metaphase I transition in mouse spermatocytes. Chromosoma 117: 471–485. 13. An, W. F., M. R. Bowlby, M. Betty, J. Cao, H. P. Ling, G. Mendoza, 36. Hosokawa, N., I. Wada, K. Hasegawa, T. Yorihuzi, L. O. Tremblay, A. Herscovics, J. W. Hinson, K. I. Mattsson, B. W. Strassle, J. S. Trimmer, and K. J. Rhodes. and K. Nagata. 2001. A novel ER alpha-mannosidase-like protein accelerates ER- 2000. Modulation of A-type potassium channels by a family of calcium sensors. associated degradation. EMBO Rep. 2: 415–422. Nature 403: 553–556. 37. Oda, Y., N. Hosokawa, I. Wada, and K. Nagata. 2003. EDEM as an acceptor of 14. Lilliehook, C., S. Chan, E. K. Choi, N. F. Zaidi, W. Wasco, M. P. Mattson, and terminally misfolded glycoproteins released from calnexin. Science 299: 1394– J. D. Buxbaum. 2002. Calsenilin enhances apoptosis by altering endoplasmic 1397. reticulum calcium signaling. Mol. Cell. Neurosci. 19: 552–559. 38. Peng, S. L., A. J. Gerth, A. M. Ranger, and L. H. Glimcher. 2001. NFATc1 and 15. Rivas, M., B. Mellstro¨m, J. R. Naranjo, and P. Santisteban. 2004. Transcriptional NFATc2 together control both T and B cell activation and differentiation. Im- repressor DREAM interacts with thyroid -1 and regulates munity 14: 13–20. thyroglobulin gene expression. J. Biol. Chem. 279: 33114–33122. 39. Gallo, E. M., K. Cante´-Barrett, and G. R. Crabtree. 2006. Lymphocyte calcium 16. Cheng, H. Y., G. M. Pitcher, S. R. Laviolette, I. Q. Whishaw, K. I. Tong, signaling from membrane to nucleus. Nat. Immunol. 7: 25–32. L. K. Kockeritz, T. Wada, N. A. Joza, M. Crackower, J. Goncalves, et al. 2002. 40. Wilker, P. R., M. Kohyama, M. M. Sandau, J. C. Albring, O. Nakagawa, DREAM is a critical transcriptional repressor for pain modulation. Cell 108: 31–43. J. J. Schwarz, and K. M. Murphy. 2008. Transcription factor Mef2c is required 17. Vandermeeren, A. M., C. E. Go´mez, C. Patin˜o, E. Domingo-Gil, S. Guerra, for B cell proliferation and survival after antigen receptor stimulation. Nat. J. M. Gonza´lez, and M. Esteban. 2008. Subcellular forms and biochemical events Immunol. 9: 603–612.