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Stage-specific control of early B cell development by the transcription factor Ikaros

Nature Immunology volume 15, pages 283–293 (2014)Cite this article

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Abstract

The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell–specific function by conditional inactivation of the gene encoding Ikaros (Ikzf1) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.

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Figure 1: Ikaros loss in pro-B cells arrests development at the transition from pro-B cell to pre-B cell.
Figure 2: Cell-cycle arrest of Ikaros-deficient c-Kitlo 'pro-B cells' despite immunoglobulin μ-chain expression.
Figure 3: Identification of regulated Ikaros targets in pro-B cells.
Figure 4: Function of proteins encoded by activated and repressed Ikaros targets in pro-B cells.
Figure 5: Ikaros controls pre-BCR signaling and the migration and adhesion of cells.
Figure 6: Overlap of Ikaros binding with the regulatory 'landscape' and other transcription factor–binding sites in pro-B cells.
Figure 7: Chromatin changes at promoters and distal elements of Ikaros-regulated genes.
Figure 8: Ikaros regulates distinct target genes during early B lymphopoiesis.

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Acknowledgements

We thank G. Schmauß, T. Lendl and G. Stengl for sorting cells by flow cytometry; C. Theussl and J. Wojciechowski for blastocyst injection; B. Vilagos for the EBF1 ChIP-Seq data; K. Georgopoulos (Harvard University) for the Ikzf1+/− mouse and mouse mAb 4E9 to Ikaros; H. Karasuyama (Tokyo Medical and Dental University) for the antibody to CD79b; and A. Sommer and his team at the Campus Science Support Facilities for Illumina sequencing. Supported by Boehringer Ingelheim, the European Community's Seventh Framework Programme (European Research Council Advanced Grant 291740-LymphoControl), the Austrian GEN-AU initiative (financed by the Bundesminsterium für Bildung und Wissenschaft) and the European Molecular Biology Organization (T.A.S.).

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Author notes

  1. Aleksandar Dakic

    Present address: Present address: The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.,

  2. Tanja A Schwickert and Hiromi Tagoh: These authors contributed equally to this work.

Authors and Affiliations

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

    Tanja A Schwickert, Hiromi Tagoh, Sinan Gültekin, Aleksandar Dakic, Elin Axelsson, Martina Minnich, Anja Ebert, Barbara Werner, Mareike Roth, Johannes Zuber, Markus Jaritz & Meinrad Busslinger

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

    Luisa Cimmino & Ross A Dickins

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Contributions

T.A.S. and H.T. did most experiments; S.G. analyzed Ikzf1ihCd2/+ mice and generated the Ikaros ChIP-Seq data of progenitor cells with Ebf1 mutation; E.A. and M.J. did the bioinformatics analysis of all RNA-Seq and ChIP-Seq data, respectively; M.M. and A.E. provided the PU.1 and IRF4 ChIP-Seq data, respectively; A.D. and B.W. generated the Ikzf1ihCd2/+ and Ikzf fl/fl mice, respectively; L.C. and R.A.D. characterized the Ikzf1-specific shRNAs; M.R. and J.Z. provided advice and help with the shRNA experiments; and T.A.S., H.T. and M.B. planned the project, designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Meinrad Busslinger.

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Integrated supplementary information

Supplementary Figure 1 Generation, characterization and expression of the Ikzf1ihCd2 allele.

(a) Structure of the Ikzf1ihCd2 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 Ikzf1 gene. Brackets indicate the two homology regions mediating recombination in ES cells. The last Ikzf1 exons are shown as open boxes. LoxP and frt sites are indicated by red and yellow arrowheads, respectively. The XbaI (X) fragments of the wild-type and Ikzf1ihCd2-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 Ikzf1 codon contained cleavage sites for the PreScission (PreSc) and TEV proteases, epitopes for Flag and V5 antibodies and a biotin acceptor sequence (Biotin) for biotinylation by the E. coli biotin ligase BirA54. pA, polyadenylation site. (b) PCR identification of the Ikzf1ihCd2 allele in tail DNA from mice of the indicated genotypes. (c) Efficient interaction of the Ikaros-Bio protein with streptavidin. Nuclear lysates of Ikzf1ihCd2/+ Rosa26BirA/BirA (Bio/+) and wild-type (+/+) pro-B cells were heated in 1x SDS sample buffer, cooled down prior to the addition of streptavidin (SA) and then separated by SDS-PAGE followed by immunoblotting with Ikaros antibodies. (d) Normal hematopoiesis in Ikzf1ihCd2/ihCd2 Rosa26BirA/BirA mice. Bone marrow, spleen and thymus from Ikzf1ihCd2/ihCd2 Rosa26BirA/BirA (black bars) and control Ikzf1ihCd2/+ Rosa26BirA/BirA (grey bars) mice at the age of 7 weeks were analyzed by flow cytometry, and absolute cell numbers with standard error of the mean (SEM) are shown for the indicated cell types as defined in the Online Methods. Six mice were analyzed per genotype. (e) Ikaros expression during T cell development. The thymus of Ikzf1ihCd2/+ (grey) and wild-type (black line) mice was analyzed by flow cytometry for human (h) CD2 expression at different CD4 CD8 double-negative (DN), double-positive (DP) and single-positive (SP) stages, which were defined as described in the Online Methods. The data are representative of two independent experiments. (f) Ikaros protein expression was analyzed by intracellular staining of the indicated B cell types from Ikzf1Δ/+ (grey) and Ikzf1Δ/− (back line) mice. The pro-B cell staining was controlled with pro-B cells of Cd79a-Cre Ikzf1fl/− mice, whereas CD19+ bone marrow cells of the same mice (dashed line) served as controls for pre-B and immature B cells. Cd23-Cre Ikzf1fl/− mice were used to control the staining of splenic FO, MZ and B-1 B cells. The data are representative of two independent experiments. (g) Correlation of hCD2 reporter and Ikaros protein expression in granulocytes (Gran), macrophages (Mac) and pre-B cells of Ikzf1ihCd2/ihCd2 mice, as shown by cell surface hCD2 and intracellular Ikaros staining. The data are representative of two independent experiments.

Supplementary Figure 2 Generation and characterization of the loxP-flanked Ikzf1 allele.

(a) Structure of the targeted Ikzf1 allele. The last exons of Ikzf1 are shown as open boxes. Brackets indicate the two homology regions mediating recombination in ES cells. LoxP and frt sites are indicated by red and yellow arrowheads, respectively. The KpnI (K) fragments, which are indicative of the wild-type, Ikzf1fl-Neo and Ikzf1fl alleles, are shown together with their length (in kb). (b) Southern blot analysis. KpnI-digested tail DNA from mice of the indicated genotypes was analyzed by hybridization with the DNA probe shown in (a). The size of the DNA fragments is indicated in kb to the right. (c) PCR genotyping of tail DNA isolated from mice of the indicated genotypes. The size of the PCR fragments is indicated in base pairs to the right. (d) Normal B cell development in Ikzf1fl/fl mice. Bone marrow and thymus from wild-type mice (white bars), Ikzf1fl/+ (grey bars) and Ikzf1fl/fl mice (black bars) at the age of 8-9 weeks were analyzed by flow cytometry, and the absolute cell numbers with SEM are shown for the indicated cell types, which were defined as described in the Online Methods. Six mice were analyzed per genotype. (e) Absence of a haploinsufficient phenotype in adult Ikzf1+/− mice. The indicated hematopoietic cell types were examined by flow cytometric analysis of the bone marrow and thymus from Ikzf1fl/+ (grey bar) and Ikzf1fl/− (black bar) mice at the age of 9 weeks. Absolute cell numbers with SEM are shown for the indicated cell types. Three mice were analyzed per genotype. (f) Kaplan-Meyer survival analysis of mice of the indicated genotypes. The Il7r-Cre and Rag2-Cre lines result in Cre-mediated deletion of the floxed Ikzf1 allele in uncommitted lymphoid and T lineage progenitors. As a consequence, the Ikzf1fl/− mice carrying Il7r-Cre or Rag2-Cre died of aggressive T cell leukemia (identified by flow cytometric analysis), similar to Ikzf1−/− mice2. n indicates the number of mice analyzed.

Supplementary Figure 3 In vitro culture of Ikzf1Δ/− pro-B cells and in vivo phenotype Ikzf1Δ/− Rag2−/− pro-B cells.

(a) Flow cytometric analysis of bone marrow pro-B cells of the indicated genotypes, which were cultured for 7 days in the presence of IL-7 and stromal OP9 cells. Pro-B cells were FACS-sorted as c-KithiCD19+CD2−CD5−IgM−IgD− cells prior to RNA preparation and sequencing. The data are representative of four independent experiments. (b) PCR analysis. Deletion of the floxed Ikzf1 allele was analyzed by PCR in short-term cultured pro-B cells of Cd79a-Cre Ikzf1fl/− and control Ikzf1fl/− mice. PCR fragments corresponding to the deleted (Δ) or intact (fl) floxed allele are indicated to the left and their size (in base pairs) to the right of the PCR gel. (c) Loss of Ikaros protein. Nuclear extracts of in vitro-cultured Cd79a-Cre Ikzf1fl/− (Ikzf1Δ/−) and Cd79a-Cre Ikzf1fl/+ (Ikzf1Δ/+) pro-B cells were analyzed by immunoblotting with Ikaros and TBP antibodies. The positions of the Ikaros isoforms 1 and 2 as well as of molecular size markers (in kilodaltons) are indicated to the left and right of the immunoblot, respectively. (d,e) Cell cycle analysis. In vitro-cultured Ikzf1Δ/− and Ikzf1Δ/+ pro-B cells were analyzed for their cell size (d) and relative distribution in the different cell cycle phases (e) based on flow cytometric measurement of DNA content (7-AAD) and BrdU incorporation, as described in the Online Methods. The data are representative of two independent experiments. (f,g) Flow cytometry (f) and relative percentage (g) of the indicated pro-B cells in the bone marrow of Cd79a-Cre Ikzf1fl/+ Rag2−/− mice (grey bars; Δ/+) and Cd79a-Cre Ikzf1fl/− Rag2−/− littermates (black bars; Δ/−). Four mice of each genotype were analyzed. Numbers refer to percent cells in the indicated gate (f). (h) Cell cycle analysis. Total and c-Kithi pro-B cells of Cd79a-Cre Ikzf1fl/+ Rag2−/− (grey bars; Δ/+) and Cd79a-Cre Ikzf1fl/− Rag2−/− (black bars; Δ/−) mice were analyzed for their distribution in the different cell cycle phases, as described in the Online Methods. Three mice of each genotype were analyzed. Statistical data are shown with SEM and were analyzed by two-way analysis of variance (ANOVA) with Bonferoni's post-tests; * (P < 0.05), ** (P < 0.01), *** (P < 0.001).

Supplementary Figure 4 Flow cytometry sorting and Ikzf3 expression in Ikzf1 mutant pro-B cells.

(a) FACS sorting of c-KithiCD19+CD25−IgM−IgD− pro-B cells from bone marrow of the indicated genotypes at the age of 4-8 weeks. The different gates used for FACS sorting (left) are indicated. The purity of the sorted cell population was determined by flow cytometric reanalysis (right). The cells in the different gates are shown as relative percentages. (b) Absence of the last exon in Ikzf1 transcripts following gene inactivation. The RNA-seq profile of the Ikzf1 gene is shown for in vitro short-term cultured and ex vivo-sorted pro-B cells from the bone marrow of Cd79a-Cre Ikzf1fl/− (Ikzf1Δ/−) and Cd79a-Cre Ikzf1fl/+ (Ikzf1Δ/+) mice. Exon 8 is highlighted by yellow shading. Ikaros-binding sites were identified by Bio-ChIP-sequencing. (c) Quantification of exon 8 transcript levels. Ikzf1 transcript reads corresponding to the exon 8 sequences (mm9 Chr. 11, 11668880-11672178), which are deleted both in the null (−) and deleted (Δ) Ikzf1 allele, are shown as RPKM values. (d,e) Activation of the Ikzf3 (Aiolos) gene in Ikaros-deficient pro-B cells. The RNA-seq profiles of the Ikzf3 gene in short-term in vitro-cultured and ex vivo-sorted Ikzf1Δ/− and Ikzf1Δ/+ pro-B cells are shown together with Ikaros-binding sites identified in pro-B cells by Bio-ChIP-sequencing (d). Ikzf3 expression in the different pro-B cell types is shown as average RPKM value (plus SEM) of two RNA-seq experiments (e). The data shown in (b-d) are based on two independent RNA-seq experiments per cell type.

Supplementary Figure 5 Ikaros-regulated genes encoding molecules with functions linked to pre-BCR signaling, cell adhesion and migration.

The expression of the indicated genes was determined by RNA-sequencing of (i) in vitro-cultured Cd79a-Cre Ikzf1fl/+ (Ikzf1Δ/+) Rag2–/– (1) and Cd79a-Cre Ikzf1fl/– (Ikzf1Δ/–) Rag2–/– (2) pro-B cells, (ii) ex vivo-sorted Ikzf1Δ/+ (3) and Ikzf1Δ/– (4) c-Kithi pro-B cells as well as (iii) the in vitro-cultured Ikzf1Δ/+ (5) and Ikzf1Δ/– (6) pro-B cells that were used to define regulated Ikaros target genes in this study (Fig. 3f,g; Supplementary Table 1). The expression of each gene in Ikzf1Δ/+ (grey bar) and Ikzf1Δ/– (black bar) pro-B cells is shown as normalized expression value (RPKM) with SEM based on two independent RNA-seq experiments for each genotype. (a,b) Expression of Ikaros-regulated genes with functions in pre-BCR signaling (a) or cell adhesion and migration (b). Regulated Ikaros target genes (Ikaros binding; Fig. 3f,g) and indirectly Ikaros-regulated genes (no Ikaros binding) are shown. Asterisks indicate those genes, which were not regulated (< 1.4-fold) in short-term cultured Ikzf1Δ/+ Rag2–/– (1) and Ikzf1Δ/– Rag2–/– (2) pro-B cells. (c) Expression of non-regulated genes with functions in pre-BCR signaling. This class contains genes, which were regulated by Ikaros less than the 3-fold cutoff used for identifying Ikaros-regulated genes in pro-B cells (Fig. 3f,g). (d) Other genes. This class contains genes coding for transcription factors, cell cycle regulators and effector proteins of immunoglobulin V(D)J recombination (Rag1, Rag2, TdT [Dntt], ligase 4, Ku70 [Xrcc6], DNA-PKCS [Rrkdc]). Different colors indicate genes of distinct functional categories. GEF, guanine nucleotide-exchange factor; GAP, GTPase-activating protein.

Supplementary Figure 6 Colocalization of Ikaros-binding sites with peaks of other transcription factors at selected Ikaros target genes in pro-B cells.

(a) Binding of the transcription factors Pax5, EBF1, PU.1, IRF4 and Ikaros is shown at 15 Ikaros target genes, which were activated, repressed or not regulated by Ikaros in pro-B cells (Fig. 3f,g). Pro-B cells were analyzed by ChIP-seq to identify binding sites of Pax5 (ref. 20), EBF1 (ref. 26), PU.1 (see Online Methods) and IRF4 (see Online Methods) in addition to Ikaros (Fig. 3a,b). Bars below the ChIP-seq track indicate binding regions, which were called by the MACS program with a stringent P-value of < 10−10. The exon-intron structure of each gene is shown together with a black scale bar indicating one kb. Grey shading indicates the colocalization of Pax5- and Ikaros-binding sites at regulatory elements of Id3 and Cst7. (b) Coregulation of genes in pro-B cells by the transcription factors Ikaros and Pax5. Genes were only considered for this analysis, if one of their regulatory elements was simultaneously bound by Pax5 and Ikaros (Fig. 6d) and was also regulated by both transcription factors in pro-B cells, as shown for Id3 and Cst7 in (a) and (b). The expression of each gene in cultured Ikzf1Δ/+ (grey) and Ikzf1Δ/− (black) pro-B cells (Fig. 3f,g) as well as in cultured wild-type (light blue) and Pax5Δ/Δ (dark blue) pro-B cells20 is shown as RPKM value with SEM based on two independent RNA-seq experiments for each genotype.

Supplementary Figure 7 Mi-2β binding to Ikaros peaks at promoters and distal elements in the presence and absence of Ikaros.

(a) Number and overlap of Ikaros and Mi-2β (CHD4) peaks at promoters and distal elements in pro-B cells. The total numbers of Ikaros and Mi-2β peaks with the indicated overlap (black bar) were identified by ChIP-seq in short-term cultured Rag2−/− and Cd79a-Cre Ikzf1fl/+ (Ikzf1Δ/+) pro-B cells, respectively, by using a P-value of < 10-10 for peak calling. (b) The densities of Ikaros and Mi-2β binding at promoters and enhancers are shown as heat maps for pro-B cells of the indicated genotypes. The pro-B cells used for determining the H3K9ac pattern were additionally Rag2-deficient. The regulatory regions extend from -2.5 kb to +2.5 kb relative to the TSS (promoters) or the center of the DHS sites (distal elements) and were sorted according to increasing density of the Ikaros peaks. (c) Quantification of Mi-2b binding at promoter and distal elements containing Ikaros peaks. The Ikaros target genes were classified according to their degree of Ikaros-dependent regulation. The average density of Mi-2b binding in Ikzf1Δ/+ (blue) and Ikzf1Δ/− (red) pro-B cells is shown for a region extending from -2.5 kb to +2.5 kb relative to the TSS at promoters or center of the DHS site at distal elements. Only promoters and distal elements of genes with an expression level of > 2 RPKM in control pro-B cells (activated, not regulated) or in Ikaros-deficient pro-B cells (repressed) were analyzed. The number of peaks analyzed is shown for each category. The Ikaros and Mi-2β ChIP-seq experiments were performed once, and the H3K9ac ChIP experiment twice for each genotype.

Supplementary Figure 8 Tetracycline-regulated expression of Ikzf1-specific shRNA.

(a) Developmental regulation of Ikaros-repressed target gene with known functions in cell adhesion and migration. The expression of each gene in wild-type BLP, pro-B and pre-B cells is shown as normalized expression value (RPKM) with SEM based on two independent RNA-seq experiments for each cell type. Only the repressed genes (listed in Supplementary Fig. 5b), which were expressed at an RPKM value of > 5 in one of the three cell types, are shown. (b) Position of the target sequences for the two Ikzf1 (Ik) shRNA in the 3′ UTR of the mouse Ikzf1 gene. (c) Schematic diagram of the dual-color retroviral vector TRMPVIR (TRE-dsRed-miR30/shRNA-PGK-Venus-IRES-rtTA)29 containing the optimized miR-E backbone30 for efficient Tet-on induction of Ikzf1 shRNA expression. The phosphoglycerate kinase (PGK) promoter drives constitutive expression of the Venus and reverse tetracycline transactivator (rtTA3) genes. Induction of rtTA3 activity by doxycycline (Dox) addition activates the tetracycline-response element (TRE) promoter that gives rise to strong expression of a transcript containing the dsRed-shRNA gene cassette. Retrovirally infected cells that express Ikzf1 shRNA upon Dox treatment can be identified and sorted as Venus+dsRed+ cell by flow cytometry. IRES, internal ribosome entry site. (d) Analysis of shRNA-mediated down-regulation of Ikaros expression by intracellular Ikaros staining. Retrovirally infected and Dox-treated Ebf1−/− progenitor cells, expressing the indicated Ikzf1 shRNAs, Renilla luciferase Ren713 shRNA or empty TRMPVIR vector (control), were analyzed by intracellular staining with a rabbit polyclonal Ikaros antibody (black line) or control IgG (grey) by gating on infected Venus+dsRed+ or non-infected Venus−dsRed− cells. (e) Immunoblot analysis of Ikaros expression. Venus+dsRed+ or Venus−dsRed− cells after the indicated treatment were FACS-sorted prior to whole cell extract preparation and immunoblotting with Ikaros and TBP antibodies. The positions of the Ikaros isoform 1 and 2 are indicated. The experiments shown in (d,e) were performed once. (f) Correlation of gene expression differences detected between Ebf1−/− progenitor cells expressing Ik4056 or Ik2709 shRNA. The fold expression change of each Ikaros-activated (blue) or Ikaros-repressed (red) gene, which was identified by comparison of Ik4056 shRNA-expressing and control Ebf1−/− progenitor cells in Fig. 8e, is shown on the y-axis and is compared to the corresponding expression change observed between Ik2709 shRNA-expressing and control Ebf1−/− progenitor cells on the x-axis.

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Supplementary Figures 1–8 and Supplementary Table 2 (PDF 5651 kb)

Supplementary Table 1

Repressed and activated Ikaros target genes in pro-B cells cultured short-term or sorted ex vivo. (XLSX 196 kb)

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Schwickert, T., Tagoh, H., Gültekin, S. et al. Stage-specific control of early B cell development by the transcription factor Ikaros. Nat Immunol 15, 283–293 (2014). https://doi.org/10.1038/ni.2828

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