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Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation

Nature Immunology volume 17, pages 331–343 (2016)Cite this article

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Abstract

The transcription factor Blimp-1 is necessary for the generation of plasma cells. Here we studied its functions in plasmablast differentiation by identifying regulated Blimp-1 target genes. Blimp-1 promoted the migration and adhesion of plasmablasts. It directly repressed genes encoding several transcription factors and Aicda (which encodes the cytidine deaminase AID) and thus silenced B cell–specific gene expression, antigen presentation and class-switch recombination in plasmablasts. It directly activated genes, which led to increased expression of the plasma cell regulator IRF4 and proteins involved in immunoglobulin secretion. Blimp-1 induced the transcription of immunoglobulin genes by controlling the 3′ enhancers of the loci encoding the immunoglobulin heavy chain (Igh) and κ-light chain (Igk) and, furthermore, regulated the post-transcriptional expression switch from the membrane-bound form of the immunoglobulin heavy chain to its secreted form by activating Ell2 (which encodes the transcription-elongation factor ELL2). Notably, Blimp-1 recruited chromatin-remodeling and histone-modifying complexes to regulate its target genes. Hence, many essential functions of plasma cells are under the control of Blimp-1.

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Figure 1: Blimp-1-dependent gene-expression changes during plasma cell differentiation.
Figure 2: Identification of target genes regulated by Blimp-1.
Figure 3: Function of activated and repressed Blimp-1 target genes in plasmablasts.
Figure 4: Mutually exclusive binding of Blimp-1 and the PU.1-IRF4 complex to a common recognition motif in plasmablasts.
Figure 5: Blimp-1-dependent regulation of transcription factor–encoding genes in plasmablasts.
Figure 6: Blimp-1-dependent activation of immunoglobulin gene transcription in plasmablasts.
Figure 7: Chromatin changes at regulated Blimp-1 target genes in plasmablasts.
Figure 8: Blimp-1-dependent recruitment of chromatin modifiers and remodelers to target genes.

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Acknowledgements

We thank D. Kostanova Poliakova for technical assistance; K. Aumayr and colleagues for sorting by flow cytometry; K. Mechtler and colleagues for mass spectrometry; A. Sommer and colleagues for Illumina sequencing at the Vienna Biocenter Core Facility; H. Ekker for bioinformatics processing of ATAC-seq data; R. Kingston (Massachusetts General Hospital) for antibody to Brg1; and T. Jenuwein (Max Planck Institute of Immunobiology and Epigenetics, Freiburg) for antibody to H3K27me3. Supported by Boehringer Ingelheim, the European Research Council (European Union Seventh Framework Program: 291740-LymphoControl), the Austrian Industrial Research Promotion Agency, the Emerald Foundation (Tarakhovsky laboratory), the National Health and Medical Research Council of Australia (361646 and 1054925 for the Nutt laboratory) and the Multiple Myeloma Research Foundation (for the Nutt laboratory).

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  1. Elin Axelsson

    Present address: Present address: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.,

Authors and Affiliations

  1. Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria

    Martina Minnich, Hiromi Tagoh, Peter Bönelt, Elin Axelsson, Maria Fischer, Beatriz Cebolla, Markus Jaritz & Meinrad Busslinger

  2. Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, New York, USA

    Alexander Tarakhovsky

  3. The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

    Stephen L Nutt

  4. Department of Medical Biology, The University of Melbourne, Parkville, Australia

    Stephen L Nutt

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Contributions

M.M. did most experiments; H.T. performed the ATAC-seq, IRF4 ChIP-seq, electrophoretic mobility-shift assay and Blimp-1–ER–induction experiments; P.B. performed the migration, adhesion and HEK-293T cell co-precipitation experiments; E.A. and M.J. did the bioinformatics analysis of all RNA-seq data and ChIP-seq data, respectively; M.F. performed the bioinformatics analysis of the immunoglobulin-encoding transcripts; B.C. generated the targeted Prdm1ihCd2-Neo embryonic stem cells; A.T. and S.L.N. provided advice and important mouse strains; and M.M., H.T. and M.B. planned the project, designed the experiments and wrote the manuscript.

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Correspondence to Meinrad Busslinger.

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Integrated supplementary information

Supplementary Figure 1 Blimp-1-dependent gene-expression changes during plasmablast differentiation.

(a) Flow cytometric sorting of activated B cells (Act B; CD22+CD138−GFP−), pre-plasmablasts (pre-PB; CD22−CD138−GFP+) and plasmablasts (PB; CD22−CD138+GFP+) after 4 days of LPS stimulation of B220+ mature B cells, which were isolated by immunomagnetic sorting from the spleen and lymph nodes of Prdm1Gfp/+ mice. The purity of the sorted cells was determined by flow cytometric reanalysis. The reanalysis of each of the three individual cell populations is shown in a different color in one of the three quadrants (combined from three FACS plots) together with the corresponding purity (indicated as percent of cells). (b) Scatter plot of gene expression differences observed between pre-plasmablasts and activated B cells (left) or pre-plasmablasts and plasmablasts (right) of the Prdm1Gfp/+ genotype. Genes in these two cell type comparisons are colored in blue or red, if they correspond to up-regulated (blue) or down-regulated (red) genes identified in the activated B cell-to-plasmablast transition (plasmablast signature; Fig. 1b, Supplementary Table 1), respectively. (c) Flow cytometry sorting and reanalysis of plasma cells (B220loCD28+CD138+GFP+Lin−) from the bone marrow of unimmunized Prdm1Gfp/+ mice at the age of 6 months. (d) PCR genotyping of FACS-sorted activated B cells (Act B; CD138−GFP−), pre-plasmablasts (pre-PB; CD138−GFP+, top row) and plasmablasts (PB; CD138+GFP+). The pre-plasmablasts were further separated into CD22loCD138−GFP+ and CD22hiCD138−GFP+ cells (right). The positions of the PCR fragments corresponding to the Gfp-tagged, intact floxed (fl) and deleted (∆) Prdm1 alleles are shown to the left, and their size is indicated in base pairs (bp) to the right. (e) Flow cytometric sorting and reanalysis of Prdm1-deleted activated B cells (Act B; CD22+CD138−GFP−) and pre-plasmablasts (pre-PB; CD22+CD138−GFP+) after 4 days of in vitro LPS stimulation of B220+ mature B cells, which were isolated by immunomagnetic sorting from the spleen and lymph nodes of Cd23-Cre Prdm1Gfp/fl mice. (f) Expression of Blimp1-dependent cell surface receptor genes during LPS-induced plasmablast differentiation and in ex vivo sorted mature B cells (Mat B) and plasma cells (PC). The expression of each gene is shown as normalized gene-expression RPKM value with SEM, based on two RNA-seq experiments for each cell type. Pre-PB, pre-plasmablasts; KO Pre-PB, Prdm1Gfp/∆ Pre-PB. (g) Expression of Blimp1-independent genes during LPS-induced plasmablast differentiation as well as in mature B cells and plasma cells. Blimp1-independent genes were defined by a < 1.6 fold expression difference between Prdm1Gfp/∆ and control Prdm1Gfp/+ pre-plasmablasts (see Fig. 1g).

Supplementary Figure 2 Generation and characterization of the Prdm1Bio allele.

(a) Structure of the Prdm1Bio allele. A C-terminal tag sequence, an IRES-hCd2 (ihCd2) reporter gene and a neomycin (neor) resistance gene under the control of the mouse phosphoglycerate kinase (Pgk1) promoter were inserted between the last codon and the 3’ untranslated region (3’UTR) of the Prdm1 gene. Brackets indicate the two homology regions mediating recombination in ES cells. Prdm1 exons are shown as open boxes, and loxP and frt sites as red and blue arrowheads, respectively. The EcoRI (E) fragments of the wild-type (Prdm1+) and Prdm1ihCd2-Neo alleles, which were used for allele identification by Southern blot analysis with the indicated probe, are shown together with their length (in kilobases, kb). The tag sequences added at the last Prdm1 codon contained epitopes for Flag and V5 antibodies, two cleavage sites for the TEV protease and a biotin acceptor sequence (Biotin) for biotinylation by the E. coli biotin ligase BirA. pA, polyadenylation site. The Prdm1ihCd2 and Prdm1Bio alleles were generated by sequential Cre- and Flpe-mediated recombination. (b) Southern blot analysis of correctly targeted (Neo/+) and wild-type (+/+) ES cell clones by hybridization of EcoRI-digested DNA with the DNA probe shown in a. (c) Efficient precipitation of the in vivo biotinylated Blimp1-Bio protein by streptavidin pulldown. Plasmablasts were generated from mature B cells of Prdm1+/+ Rosa26BirA/+ (+/+) and Prdm1Bio/Bio Rosa26BirA/+ (Bio/Bio) mice by LPS stimulation for 4 days prior to nuclear extract preparation and precipitation of the biotinylated Blimp1-Bio protein with streptavidin beads. The input (1/10), supernatant (1/10) and streptavidin-bound precipitate were analyzed by immunoblotting with an anti-Blimp1 or anti-V5 antibody. (d) Normal B cell development in Prdm1Bio/Bio mice. Bone marrow and spleen of Prdm1Bio/+ Rosa26BirA/+ (grey bars) and Prdm1Bio/Bio Rosa26BirA/BirA (black bars) mice at the age of 7 weeks were analyzed by flow cytometry, and absolute cell numbers with the standard error of the mean (SEM) are shown for pro-B (CD19+c-Kit+CD25−IgM−IgD−), pre-B (CD19+CD25+c-Kit−IgM−IgD−), immature B (CD19+IgMhiIgDlo), recirculating B (CD19+ IgMlo IgDhi), total B cells (CD19+B220+), granulocytes (Gr1hiMac1hi), erythrocytes (c-Kit−Ter119+), macrophages (Mf; Mac1+MCSF-R+). Three mice were analyzed per genotype. (e,f) Normal immune response of Prdm1Bio/Bio mice. Prdm1Bio/+ and Prdm1Bio/Bio mice were immunized with 4-hydroxy-3-nitrophenylacetyl-conjugated keyhole limpet hemocyanin (NP-KLH in Alum). At day 14 after immunization, the spleen and bone marrow were analyzed for the presence of germinal center (GC) and memory (Mem) B cells as well as plasma cells (PC) by flow cytometry (e) and for anti-NP-IgG1 antibody-secreting cells (ASC) by ELISPOT assay (f), respectively. The numbers of anti-NP-IgG1 ASCs with SEM were determined by ELISPOT assay using NP4-BSA- or NP23-BSA-coated plates for detecting cells secreting high-affinity or total anti-NP-IgG1 antibodies, respectively. Four mice were analyzed per genotype. (g) Expression of three activated Blimp1 target genes in Prdm1Gfp/∆ (KO) pre-plasmablasts and in activated B cells, pre-plasmablasts and plasmablasts of the control Prdm1Gfp/+ genotype, as shown by RNA-seq analysis. Blimp1 peaks were identified in plasmablasts by Bio-ChIP-seq. Mzb1, Hspa (BiP) and Ssr2 code for proteins involved in ER function. (h) Blimp1-ERT2-mediated down-regulation of CD22 expression. B cells of the WEHI-Blimp1-ERT2 cell clone 194 were treated with OHT (1 μM) for 30 h followed by flow cytometric analysis of CD22 and hCD2 (reporting the retroviral Blimp1-ERT2 expression).

Supplementary Figure 3 Regulated Blimp-1 target genes with functions in different pathways.

The expression of the indicated repressed and activated Blimp1 target genes was determined by RNA-sequencing of LPS-stimulated Prdm1Gfp/∆ pre-plasmablasts (KO Pre-PB, red) and activated B cells (Act B, light grey), pre-plasmablasts (Pre-PB, grey) and plasmablasts (PB, dark grey) of the control Prdm1Gfp/+ genotype as well as ex vivo sorted wild-type lymph node B cells (Mat B, white) and bone marrow plasma cells (PC, black). Gene expression is shown as normalized expression value (RPKM) with SEM, based on two independent RNA-seq experiments for each cell type. The genes are classified according to their functions in the different pathways indicated.

Supplementary Figure 4 Binding of Blimp-1 and the PU.1-IRF4 complex to the same DNA element in plasmablasts.

(a) Multiple overlap of Blimp1, IRF4 and PU.1 binding in plasmablasts. For each sector of the Venn diagram, the number of binding sites is indicated together with the top-ranked motif identified with the indicated E-value by de novo motif discovery. (b) Heat maps of all 3,806 Blimp1-binding sites overlapping with IRF4 and PU.1 peaks in plasmablasts. The heat maps are shown for a region extending from -2.5 kb to +2.5 kb relative to the Blimp1 peak summit and were sorted according to the increasing density of Blimp1 (left) or IRF4 (left) binding. Density scale; low (grey), intermediate (red), high (yellow). (c) IRF4, PU.1 and Blimp1 binding at the repressed Blimp1 target genes Tlr9, Vrk2 and Aff3 and the non-regulated genes Cobll1 and Rps14. The ChIP-qPCR experiments shown in Fig. 4e were performed with the common binding sites (Tlr9, Vrk2, Aff3) and sites (Cobll1, Rps14) interacting only with PU.1 and IRF4, both of which are indicated by red and blue boxes, respectively. (d) Structure of the Spi1Bio allele. A C-terminal tag sequence, an ihCd2 reporter gene and a neomycin (neor) resistance gene under the control of the mouse phosphoglycerate kinase (Pgk1) promoter were inserted between the last codon and the 3’ untranslated region (3’UTR) of the Spi1 (PU.1) gene. The EcoRI (E) fragments of the wild-type (Spi1+) and Spi1ihCd2-Neo alleles, which were used for allele identification by Southern blot analysis with the indicated probe, are shown together with their length (in kilobases, kb). The tag sequences added at the last Spi1 codon contained cleavage sites for the PreScission (PreSc) and TEV proteases, epitopes for Flag and V5 antibodies and a biotin acceptor sequence (Biotin). pA, polyadenylation site. Right: Southern blot analysis of correctly targeted (Neo/+) and wild-type (+/+) ES cell clones by hybridization of EcoRI-digested DNA with the DNA probe shown in the left diagram.

Supplementary Figure 5 Blimp-1-binding patterns at regulated transcription factor–encoding genes in plasmablasts.

(a,b) Blimp1 binding at repressed (a) and activated (b) target genes coding for the indicated transcriptional regulators. Blimp1-binding was determined by Bio-ChIP-sequencing of Prdm1Bio/Bio Rosa26BirA/BirA plasmablasts after 4 days of LPS stimulation. Blimp1 peaks are shown together with the exon-intron structure of the gene and a scale bar shown in kilobases (kb). Bars below the ChIP-seq track indicate Blimp1-binding regions identified by MACS peak calling. The B cell-specific promoter (pIII) of the Ciita gene is additionally shown. (c,d) Blimp1-binding pattern at the Bcl6 (c) and Xbp1 (d) genes in plasmablasts.

Supplementary Figure 6 Blimp-1-dependent activation of the Igk locus in plasmablasts.

(a) Blimp1 binding and transcript abundance at the Igk locus. The binding of the Blimp1-Bio protein (determined by ChIP-seq; top row) in plasmablasts (PB) and the abundance of the gene transcripts (determined by RNA-seq) is shown for LPS-stimulated Prdm1Gfp/∆ pre-plasmablasts (KO Pre-PB) as well as for activated B cells (Act B), pre-plasmablasts (Pre-PB) and plasmablasts (PB) of the control Prdm1Gfp/+ genotype. The annotation of the C57BL/6 Igk locus (below) shows the large Vκ gene cluster and the 3’ proximal domain containing the Jκ and Cκ gene segments as well as the Igk enhancers. (b) Chromatin changes and transcription factor binding at the enhancers in the 3’ end of the Igk locus during the transition from activated B cells to plasmablasts. DHS sites were determined by ATAC-seq, the active histone mark H3K9ac and IRF4 binding by ChIP-seq and the binding of PU.1-Bio and Blimp1-Bio by Bio-ChIP-seq. Bars below the ChIP-seq track indicate Blimp1-binding regions identified by MACS peak calling. The iEκ, 3’Eκ and Ed enhancers are shown below together with a novel plasmablast-specific DHS site. (c) DHS sites in a gene-dense genomic region lacking regulated Blimp1 target genes. DHS sites were mapped by ATAC-seq in activated B cells (Act B), pre-plasmablasts (pre-PB) and plasmablasts (PB). KO, Knock-out (Prdm1Gfp/∆). (d) The average density of all DHS sites in control pre-plasmablasts, plasmablasts and KO pre-plasmablasts. A region extending from -2.5 kb to +2.5 kb relative to the center of the DHS site is shown.

Supplementary Figure 7 Interaction of Blimp-1 with the epigenetic landscape of plasmablasts.

(a) Flow cytometry sorting of activated B cells (B220+GFP−) and plasmablasts (B220intGFP+) after 4 days of LPS stimulation of spleen and lymph node B cells from Prdm1Gfp/+ mice. The purity of the sorted cells was determined by flow cytometric reanalysis and is indicated in percent of cells. (b) Definition of the epigenetic landscape of activated B cells (Act B) and plasmablasts (PB) by determining the patterns of the indicated 4 histone modifications (by ChIP-seq), DHS sites (by ATAC-seq) and CpG islands (by CAP-seq of blood cells; Illingworth et al., PLoS Genet. 6, e1001134). The densities of Blimp1 binding, DHS sites, the 4 histone marks and CpG islands (CGI) are displayed for all 8,742 Blimp1-binding sites in a region extending from -10 kb to +10 kb relative to the Blimp1 peak summit. ‘Active’ promoters (DHS sites plus annotated TSS), ‘active’ distal elements (DHS sites without TSS) and ‘inaccessible’ regions (without DHS sites without TSS), which were defined in plasmablasts, were further subdivided according to the different combinations of the histone marks. The heat maps were sorted according to the increasing density of H3K4me3 in plasmablasts. Density scale; low (grey), intermediate (red), high (yellow). (c) No increase of H3K27me3 at genes that were repressed (> 3x), but lacked Blimp1-binding sites in plasmablasts. The average density of H3K27me3 in activated B cells (light blue) and plasmablasts (dark blue) is shown for a region extending from -2.5 kb to +2.5 kb relative to the center of DHS site at these genes. (d) Absence of H3K27me3 at Blimp-binding sites of repressed target genes in Blimp1-deficient pre-plasmablasts. H3K27me3 was mapped by ChIP-seq analysis of Prdm1Gfp/∆ pre-plasmablasts (KO pre-PB; CD138−GFP+CD22+) and control Prdm1Gfp/+ pre-plasmablasts (pre-PB; CD138−GFP+CD22−), which were isolated by flow cytometric sorting after 4 days of LPS stimulation. The densities of H3K27me3 at Blimp1-binding sites of repressed target genes are shown as a density diagram corresponding to the heat map shown in Fig. 7e. (e) Presence of CpG islands (CGI) at Blimp1-bound promoters and distal elements. The overlap of CpG islands with promoters (defined by an annotated TSS) and distal elements (lacking a TSS) is shown as percentage relative to all 8,742 Blimp1-binding sites in the genome, all Blimp1-binding sites at repressed target genes (Fig. 2c) or only the Blimp1-binding sites displaying bilateral H3K27me3 spreading at repressed target genes. (f) Presence of active (H3K4me3, H3K9ac) and repressive (H3K27me3) histone marks, DHS sites, CpG islands (CGI; blood) and Blimp1-binding sites at the repressed target gene Vrk2 in activated B cells (Act B), pre-plasmablasts (pre-PB) and plasmablasts (PB). KO, Knock-out (Prdm1Gfp/∆).

Supplementary Figure 8 Partial silencing of repressed Blimp-1 target genes via inhibition of Ezh2.

(a,b) Partial silencing of repressed Blimp1 target genes by inhibition of the Ezh2 activity with the chemical compound GSK343 (Verma et al., ACS Med. Chem. Lett. 3, 1091-1096). The inhibitor GSK343 (2.5 μM) or control DMSO was added to Prdm1Gfp/+ B cells at the start of LPS-induced differentiation, followed by RNA-sequencing of sorted CD138−GFP+ pre-plasmablasts. (a) The gene expression differences between GSK343- and DMSO-treated pre-plasmablasts are shown as a scatter blot, based on two RNA-seq experiments for each treatment. The repressed Blimp1 target genes are highlighted in red. (b) Expression of selected repressed Blimp1 target genes with H3K27me3 spreading in GSK343- and DMSO-treated Prdm1Gfp/+ pre-plasmablasts. The mRNA levels of the indicated genes, which are furthermore characterized by H3K27me3 spreading in plasmablasts (Fig. 7c), are shown as normalized gene-expression RPKM values with SEM, based on two RNA-seq experiments for each treatment. (c) No recruitment of Blimp1-ERT2, HDAC1, Ezh2, CHD4, NCOR1 and Brg1 proteins to the promoter of the control Cd19 gene. WEHI-Blimp1-ERT2 B cells were treated for up to 2 h with 4-hydroxytamoxifen (OHT, 1 μM) prior to ChIP with antibodies precipitating the indicated proteins. Input and precipitated DNA were quantified by real-time PCR with primer pairs amplifying the Cd19 and control Tbp promoters. The enrichment of precipitated DNA at the Cd19 promoter relative to the Tbp promoter was determined as described in the legend of Fig. 2f. The relative enrichment at time point 0 was set to 1. Two (Blimp1-ER, Ezh2) or three (others) independent ChIP-qPCR experiments were performed.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Methods and Supplementary Tables 5–7 (PDF 2155 kb)

Supplementary Table 1

Gene expression differences between LPS stimulated Activated B cells and plasmablasts (XLSX 260 kb)

Supplementary Table 2

Gene expression differences between ex vivo mature B cells and plasma cells (XLSX 407 kb)

Supplementary Table 3

Gene expression differences between LPS stimulated WT Preplasmablasts and Blimp1 KO Pre-plasmablasts (XLSX 183 kb)

Supplementary Table 4

Activated and Repressed Blimp1 target genes identified in pre-plasmablasts (GC-normalized RPKM values) (XLSX 116 kb)

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Minnich, M., Tagoh, H., Bönelt, P. et al. Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation. Nat Immunol 17, 331–343 (2016). https://doi.org/10.1038/ni.3349

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