Precocious expression of Blimp1 in B cells causes autoimmune disease with increased self‐reactive plasma cells

• DOI: 10.15252/embj.2018100010
• Publication Date: 2018-11-29T19:15:38Z
• Authors: Peter Bönelt; Miriam Wöhner; Martina Minnich; Hiromi Tagoh; Maria Fischer; Markus Jaritz; Anoop Kavirayani; Manasa Garimella; Mikael CI Karlsson; Meinrad Busslinger
• Keywords: Ectopic expression

Article29 November 2018Open Access Transparent process Precocious expression of Blimp1 in B cells causes autoimmune disease with increased self-reactive plasma Peter Bönelt Research Institute Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Miriam Wöhner Martina Minnich Hiromi Tagoh Maria Fischer Markus Jaritz Anoop Kavirayani Core Facilities (VBCF), Manasa Garimella Department Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden Mikael CI Karlsson Meinrad Busslinger Corresponding Author [email protected] orcid.org/0000-0002-9111-9351 Information Bönelt1, Wöhner1,‡, Minnich1,‡, Tagoh1,4, Fischer1, Jaritz1, Kavirayani2, Garimella3, Karlsson3 *,1 1Research 2Vienna 3Department 4Present address: Ludwig Cancer Research, University Oxford, UK ‡These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: The EMBO Journal (2019)38:1-19https://doi.org/10.15252/embj.2018100010 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract transcription factor is not only an essential regulator cells, but also risk development humans. Here, we demonstrate mouse that Prdm1 (Blimp1) gene was partially activated at chromatin level early cell development, although mature mRNA did accumulate due posttranscriptional regulation. By analyzing model facilitated ectopic protein throughout lymphopoiesis, could impaired interfering program, while leading abundance promoting premature plasmablast differentiation immature cells. With progressing age, these mice developed characterized presence autoantibodies glomerulonephritis. Hence, data identified as novel mechanism, through which can act disease. Synopsis Ectopic (Prdm1) (Prdm1ihCd2/+) interferes efficiently differentiation. develop Transcription developing wild-type does Prdm1ihCd2/+ impairs lymphopoiesis strong increase Blimp1-expressing undergo thus potentially evade central peripheral tolerance mechanisms. suggesting may similar manner human Introduction Plasma serve important role acute response infection long-term protection host providing humoral immunity continuous secretion antibodies. Moreover, often contribute pathogenesis secreting antibodies (Suurmond Diamond, 2015; Tsokos et al, 2016). zinc finger (encoded gene) key (Nutt 2007), initially discovered its ability induce plasmacytic upon (Turner 1994). Within B-lymphoid lineage, predominantly expressed antibody-secreting where highest observed quiescent long-lived (Kallies 2004). Consistent pattern, are lost cell-specific deletion gene, demonstrating generation plasmablasts (Shapiro-Shelef 2003; Kallies 2007). furthermore required bone marrow maintain their secretory function (Tellier Interestingly, PRDM1 frequently mutated on both alleles cell-like diffuse large lymphoma (ABC-DLBCL), loss tumor-suppressor contributes lymphomagenesis preventing (Pasqualucci 2006; Tam 2006). In addition, genome-wide association studies (GWAS) have susceptibility diseases systemic lupus erythematosus (SLE) rheumatoid arthritis (RA), two informative single nucleotide polymorphisms (SNPs) been specifically mapped intergenic region between ATG5 loci SLE RA patients (Gateva 2009; Raychaudhuri Zhou 2011; Appendix Fig S1A). To date, there are, however, no functional available would causally implicate or RA. At molecular level, functions transcriptional repressor activator recruiting chromatin-remodeling histone-modifying complexes target genes (Minnich Systematic analysis regulated multiple roles coordinating 2016; Tellier For instance, promotes migration adhesion plasmablasts. It directly represses several those coding Pax5, co-activator CIITA, cytidine deaminase AID, leads silencing expression, antigen presentation class switch recombination plasmablasts, respectively (Shaffer 2002; activates genes, IRF4 proteins involved immunoglobulin Importantly, strongly induces heavy-chain (Igh) κ light-chain (Igk) regulates from membrane-bound secreted Ig Little so far known about how MicroRNAs (miR-30b,d,e miR125b) RNA-binding ZFP36L1 implicated regulation controlling decay translation binding 3′ UTR sequences lines (Gururajan 2010; Nasir 2012; Kassambara 2017). be factors IRF4, E2A, E2-2 (Sciammas Kwon Gloury regulatory landscape locus rather complex, it consists eight open regions interact promoter located up 272 kb upstream (Wöhner already transcribed nucleus, cytoplasm study effect generated resulted insertion Moloney murine leukemia virus (MoMLV) enhancer together IRES-hCd2 (ihCd2) reporter stop codon Prdm1ihCd2 allele Consequently, activated, lymphoid progenitors being pro-B, pre-B, mice. interfered normal program activating repressing many led decreased contrast, mice, consistent finding had enhanced potential vitro phenotype anti-nuclear antibodies, immune complex deposition, kidney pathology. Together, precocious cause Results transcriptionally active We previously (sites A-H) highly express 1A). investigate epigenetic status different histone modifications ATAC-seq ChIP-seq analyses pro-B do Blimp1. Unexpectedly, contained bivalent shown (H3K4me2, H3K4me3, H3K9ac) repressive (H3K27me3) marks. A, B, C were A subset sites compared (Fig Notably, D, E, F, G, H, 2016), present closed marked contrast indicate has undergone partial activation Figure 1. Partial Mapping well Open determined (Buenrostro 2013), marks ex vivo sorted (ATAC-seq H3K27me3, study), short-term cultured H3K9ac; Revilla-i-Domingo 2012), LPS-induced Table EV4). indicated 3C mm9 genomic coordinates chromosome 10 respective positions SNPs (rs658431 rs548234) indicated. RPM, reads per million sequence reads. nascent transcripts Atg5 GRO-seq RNA-seq FO (Table Strand-specific blue red, respectively. Quantification transcript levels. mean value (TPM) SEM based experiments each type, TPM, (Wagner 2012). Download figure PowerPoint during development. test hypothesis, measured levels splenic follicular global run-on sequencing (GRO-seq; 2008). As 1B C, neighboring similarly (FO) Despite rates, low amount detected types relatively high C). prevents accumulation Posttranscriptional control mediated microRNAs principally untranslated (3′ UTR) (Pasquinelli, Alternatively, AU-rich elements (AREs) UTR, deadenylation 2014). Both mechanisms Parlato 2013; Prdm1, deleted most (2,253 bp; 90%) 2,490-bp long CRISPR/Cas9-mediated mutagenesis, left one consensus ARE motif microRNA-binding site truncated Prdm1∆3′U(90) (Appendix S1B Contrary expectation, lead S1D) elevated S1E) Prdm1∆3′U(90)/∆3′U(90) S1F). conclude therefore part (90%) dispensable expression. Premature lineages Given activity asked question whether affected lineage. end, facilitating inserting 258-bp terminal repeat (LTR) 2A) contains copies 75-bp MoMLV enhancer, binds system (Speck 1990; 2A). inserted induced end S2A) 86- 58-fold pre-B counterpart 2B). RT–qPCR revealed 26- 28-fold relative 2C). 3.4- 4-fold below genotype, rate affect 2B C) Prdm1-ihCd2 still S2B). Flpe-mediated frt-flanked containing ihCd2 restored physiological lineage summary, 2. lymphocytes A. Schematic diagram (ihCD2) linked (MoMLV, green), gene. C-terminal exon 8 in-frame tag encoding Flag V5 epitopes, TEV protease cleavage sites, biotin acceptor sequence. 1990). GRE, glucocorticoid element; LV, factor-binding site; NF1, nuclear 1; polyadenylation site, pA. B. Expression (WT) independent type genotype. C. differentiated (PB) genotypes. Plasmablasts stimulation 4 days LPS. normalized ubiquitously Tbp presented (set 1). Nascent PCR-amplified primers introns EV3). Statistical analyzed Student's t-test; *P < 0.05, **P 0.01. Each dot corresponds mouse. E. Flow cytometric hCD2 surface intracellular types. Bone (red) (WT, gray) used analyze ALPs, BLPs, NK granulocytes, whereas spleen flow MZ naïve CD4 T CD8 histograms show (top row) (bottom types, defined described Supplementary Methods. Wild-type (dashed line) negative staining difference fluorescence intensity (ΔMFI) genotypes type. F. Immunoblot TATA-binding (TBP) extracts prepared Marker size (in kilodaltons) right. next protein, reporting developmental stages analysis. uncommitted (LMPPs, BLPs); marrow; reduced marginal zone (MZ) B-1 peritoneal cavity; and, expected, 2D S2C). double-negative (DN) thymocytes, double-positive (DP) natural killer (NK) absent granulocytes macrophages 2E, S2D shown). Next, investigated S2C D), was, less sensitive analysis, closely correlated pattern S2E). readily detectable DP thymocytes S2D) confirmed immunoblotting 2F). Prdm1Gfp/ihCd2 examine auto-regulate activate second Prdm1Gfp GFP S2F), Blimp1-mediated auto-regulation occur subsets. Collectively, lower degree, Impaired 2.4-fold decrease total (CD19+B220+) cytometry 3A). Surprisingly almost completely homozygous Prdm1ihCd2/ihCd2 3A), indicating further effectively disrupted While (KithiCD19+CD25−IgM−IgD−) numbers (Kit−CD19+CD25+IgM−IgD−), (CD19+IgM+IgD−), recirculating (CD19+IgMloIgD+) 3.2-, 2.5-, 8-fold, 3B). (CD19+CD21hiCD23lo) (CD19+CD21intCD23hi) B-1a (CD5+CD19+CD23−) 2.6-, 6.9-, 21-fold, respectively, S3A). defects caused apoptosis. apoptosis assays reveal death 3D S3B), exhibited 2-fold S3B). rescued Vav-Bcl2 S3C), constitutively pro-survival Bcl2 transgene all hematopoietic (Ogilvy 1999). 3. (A) (imm) (recirc) (B) gray), (red), (white) age 6-8 weeks. spleens wild-type, Prdm1ihCd2/+, Bar graphs absolute detail splenocytes S5A. D. Determination (gray) membrane asymmetry (F2N12S ratiometric dye staining) integrity (SYTOX staining). Representative plots (left) quantification dead apoptotic (apop) (right) shown. BrdU labeling 3-month-old (red dots) littermates (gray dots). percentage BrdU+ after (day 10, white bar) subsequent 15-day chase period 25; hatched without drinking water (E). (below) indicates design experiments. obtained genotype (F). above gates Data information: (A–E) ***P 0.001, ****P 0.0001. measure lifespan continuously labeled thymidine analogue bromodeoxyuridine (BrdU) prior incorporation 3E F). published (Rolink 1998), incorporated 10% 68% (CD21−CD23−B220+CD19+) F), few recruited into pool 10-day period. 21% (2.1-fold increase) first days, half them then replaced unlabeled These shortened rapid turnover spleen. moderately even S3D–F). Pr