The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7–stimulated pro–B cell cultures revealed a reduced differentiation from large pre–B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2–deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro–B cell proliferation and small pre–B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2–deficient pro–B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

Introduction

B lymphocyte development is initiated in the bone marrow. Common lymphoid progenitor cells require the combinatorial activity of multiple transcription factors in a complex gene-regulatory network (Nutt and Kee, 2007). Ikaros and PU.1 are indispensable for the primary formation of common lymphoid progenitors, while other factors, such as E2A, early B cell factor 1 (Ebf1), Pax5, and forkhead box protein 1 (Foxo1), have important roles in the B cell–specific gene expression program (Nutt and Kee, 2007; Lin et al., 2010). Foxo1 transcriptionally up-regulates IL7R expression, controlling proliferation and apoptosis of pro–B cells after IL-7 stimulation (Milne and Paige, 2006; Dengler et al., 2008; Ochiai et al., 2012). During recombination of the IgH locus, Foxo1 and Foxo3A activate recombination-activating gene proteins 1 and 2 (Rag1 and Rag2), initiating DH-to-JH rearrangements on both IgH alleles, followed by VH-to-DJH rearrangements (Herzog et al., 2009; Clark et al., 2014). After successful VDJ recombination in IL-7–responsive pro–B cells, a μ heavy chain together with the surrogate light chain forms the pre–B cell receptor (pre-BCR) and pro–B cells develop into large pre–B cells, which become desensitized to IL-7 (Marshall et al., 1998). After a clonal expansion phase (Melchers, 1995; Herzog et al., 2009), large pre–B cells develop into small pre–B cells where VJ rearrangement on the light chain locus starts and cells stop to proliferate. The transition from large to small pre–B cells is regulated by interferon regulatory factors 4 and 8 (Irf4 and Irf8), which induce Ikaros and Aiolos expression (Ma et al., 2008). Both Irfs promote light chain rearrangement and transcription, either through direct activation of Ig light chain enhancers or indirectly through attenuation of IL-7 signaling. During the attenuation of IL-7 signaling, the transcription factor Ikaros is mandatory for the differentiation of large pre–B cells to small B cells, limiting large pre–B cell expansion by directly inhibiting the G1-S transition (Joshi et al., 2014; Schwickert et al., 2014).

Apart from the Foxo1 and Irfs transcription factors, the activator protein 1 (AP-1) family belonging to the dimeric basic region-leucine zipper transcription factors has been proposed to be important for B cell function (Karin et al., 1997). Hetero- or homodimers of Jun (c-Jun, JunB, JunD) and Fos (cFos, FosB, Fra-1, Fra-2) complexes can regulate the expression of a multitude of genes, leading to regulation of cell proliferation, apoptosis, and differentiation (Liebermann et al., 1998). In B cells, increased expression of JunB, JunD, FosB, and Fra-1 was detected after the stimulation of primary B cells through the surface BCR and/or the CD40 receptor (Tilzey et al., 1991; Huo and Rothstein, 1995, 1996). Recently, Fra-1 was found to limit plasma cell differentiation and exacerbation of antibody responses in mice (Grötsch et al., 2014). In several models, Fra-2 was shown to regulate differentiation and proliferation of cells (Lawson et al., 2009; Bozec et al., 2010). Despite the similar structure between Fra-1 and Fra-2, these two proteins have distinct target genes (Eferl et al., 2004; Bozec et al., 2010). In B cells, the role of Fra-2 remains to be determined. We hypothesized that Fra-2 deletion in B cells could regulate B lymphocyte development and activation independently of Fra-1. To determine the influence of Fra-2 in the B lineage, we crossed Mb1-Cre mice (Hobeika et al., 2006) with Fra-2 floxed mice (Eferl et al., 2007). The deletion of Fra-2 severely reduced the number of B cells in bone marrow and spleen, leading to decreased basal levels of circulating Igs. Interestingly, we demonstrated that Fra-2–deficient bone marrow B cells display strong reductions of Irf4 and Foxo1 transcript levels. A genome-wide analysis of Fra-2 occupancy revealed a complex regulatory network whereby Fra-2 induces B cell proliferation and differentiation. Our data identified Fra-2 as a key regulator of Irf4 and Foxo1 and their downstream targets Ikaros, IL7R, and Rag 1 and 2.

Results

Expression of Fra-2 on B cell subsets

To determine whether Fra-2 is an important transcription factor during B cell development, we first quantified its transcript levels in B cell subsets sorted from bone marrow and spleen as well as after in vitro B cell stimulation. Fra-2 is expressed in all B cell subsets analyzed (Fig. S1 a). On one hand, its mRNA levels decreased after LPS and anti-CD40/BCR/IL-4 stimulations of marginal, follicular, and B1 B cells (Fig. S1 b). On the other hand, Fra-2 mRNA was up-regulated in pro–B cells after 3 and 6 h of IL-7 stimulation (Fig. S1 c). Therefore, to investigate Fra-2 function during B cell development, we generated B cell–specifically Fra-2 deleted mice. Mb1-Cre mice (Hobeika et al., 2006) were crossed with mice carrying Fra-2 floxed alleles (Eferl et al., 2007) to delete Fra-2 (Fra-2ΔB cell) in B lymphocytes (Fig. S1 d). A mean of 70–80% deletion of Fra-2 on the mRNA level was observed in sorted B cell subsets from bone marrow and spleen from mutant mice compared with littermate control (Fig. S1 a).

Next, we assessed whether Fra-2 regulates B cell numbers in the bone marrow and affects B cell proliferation and transcription factor expression. Indeed, flow cytometric analyses of 6-wk-old Fra-2ΔB cell mice showed a decreased number of pro, pre, immature, and mature B cells in the bone marrow (Fig. 1, a–c). To determine the cause of the decreased B cell numbers in Fra-2–deficient mice, cell cycle analysis was performed in pro– and pre–B cells from bone marrow of Fra-2ΔB cell and littermate control mice injected in vivo with BrdU. Fra-2ΔB cell pro–B cells showed a significant reduction in G0/G1 phase compared with littermate mice (Fig. 1 d). Fra-2ΔB cell pre–B cells accumulated in the G0/G1 phases of the cell cycle, with a significant reduction in the S phase when compared with pre–B cells from littermate mice (Fig. 1 d), suggesting a defect in the proliferation of Fra-2–deficient pre–B cells. Gene profiling analysis in bone marrow B cells from littermate and Fra-2ΔB cell mice revealed that mRNA levels of molecular markers essential for B cell development and proliferation, such as Ebf1, Foxo1, Irf4, Ikaros, Aiolos, IL7Ra, Rag1, and Rag2 mRNAs, were decreased, whereas no changes were detected for Pu1, SpiB, Irf8, IgII1, and VpreB mRNA levels (Fig. 1 e). Altogether, these data suggest that Fra-2 regulates early B cell proliferation and, thereby, the output of differentiated B cells from the bone marrow, by controlling the expression of essential B cell transcription factors.

Decreased spleen B cell numbers in Fra-2–deficient mice

The decreased B cell numbers in the bone marrow of Fra-2–deficient mice prompted us to characterize peripheral B cell populations in the spleen. As shown in Fig. 2 a, the percentage and number of transitional T1 and T2 B cells remained unchanged in Fra-2–deficient mice compared with littermate controls. However, marginal and follicular B cell numbers were significantly decreased in Fra-2ΔB cell spleens (Fig. 2 b). Staining of IgM and IgD showed reduced numbers of follicular and marginal-zone B cells in spleens from mutant mice compared with littermate control (Fig. 2 c). The follicular architecture of Fra-2ΔB cell spleen appeared essentially normal (Fig. 2 d). Collectively, targeted deletion of Fra-2 in B cells appears to control the number of B2 B cells. In line with reduced B cell numbers, basal IgG1, IgG2b, and IgM titers were reduced (Fig. 2 e).

Impaired in vitro transition of large pre–B cells from Fra-2ΔB cell mice to immature B cells

To determine the mechanism leading to decreased B cell numbers in Fra-2–deficient mice, pro–B cells isolated through cell sorting were stimulated with IL-7 (Milne et al., 2004). Differentiation of pro–B cells/large pre–B cells into small pre–B cells and immature B cells was significantly decreased at high IL-7 (5 ng/ml; Fig. 3, a and b). At low IL-7 concentrations (0.5 ng/ml), differentiation of Fra-2–deficient pro–B cells/large pre–B cells into small pre–B cells was comparable with littermate control cells, and development of Fra-2–deficient immature B cells was partially rescued (Fig. S2). In addition, the proliferation of primary Fra-2–deficient pro–B cells in response to 72-h stimulation with 5 ng/ml IL-7 was decreased (Fig. 3 c) and was pinpointed to a significant decrease in DNA replication compared with littermate control pro/pre–B cell cultures (Fig. 3 d). These data are in accordance with our in vivo data (Fig. 1 d), as we applied conditions under which most pro–B cells express cytoplasmic IgM and have the capability to become pre–B cells (Marshall et al., 1998). There was no effect of Fra-2 deletion on apoptosis under these conditions (Fig. 3 e). Moreover, mature mutant and littermate control B cells proliferated similarly after stimulation with LPS or CD40L/αBCR/IL-4, suggesting a specific involvement of Fra-2 in IL-7–driven proliferation (Fig. S4, a and b).

Next, molecular analyses were performed by quantitative PCR profiling. As previously found in total bone marrow B cells in vivo (Fig. 1 e), mutant pro–B cells showed decreased expression of Ebf1, Foxo1, IL7Rα, Irf4, Ikaros, Aiolos, and Rag1/2 mRNA expression compared with littermate control pro–B cells (Fig. 3 f). Reduced Foxo1 and Rag1/2 transcripts are fully compatible with both defects in proliferation and differentiation of pre–B cells (Amin and Schlissel, 2008; Dengler et al., 2008). Because Ikaros deficiency induces pronounced cellular adherence (Schwickert et al., 2014), we assessed pro–B cell adherence in the presence of CXCL2 and IL-7. Indeed, Fra-2ΔB cell cells showed twofold increased adherence compared with control pro–B cells to vascular cell adhesion protein 1 (VCAM-1)–coated surfaces (Fig. 3 g). Collectively, these results suggest that Fra-2 plays an important role in the proliferation, adhesion, and differentiation of pro– and pre–B cells, likely by regulating the expression of essential transcription factors such as Ebf1, Ikaros, Foxo1, IL7Rα, Irf4, and Rag1/2.

Genomic targeting analysis of Fra-2 in bone marrow isolated B cells

To determine the genomic binding pattern of Fra-2 in an unbiased manner, we isolated B220+ B cells from bone marrow of Fra-2ΔB cell and littermate control mice and performed chromatin immunoprecipitation (ChIP) with Fra-2 antibody. ChIP sequencing (ChIP-seq) analyses identified consensus TRE elements of AP-1/Fra-2-binding motifs in B cells (Fig. S3 a). ChIP-seq peaks identified molecular markers related to B cell development, such as Ebf1 (Fig. 4 a), and cell cycle, BCR response, or cell proliferation, such as pdk3, lyn37, and miR125-1b (Fig. S3 b and not depicted). In accordance with Fra-2 binding to these specific targets genes, the mRNA levels of miR125-1b, lyn37, and pdk3 were decreased in Fra-2ΔB cell B cells compared with littermate controls (Fig. 4 b), suggesting that Fra-2 regulates B cell proliferation by a direct transcriptional activation of multiples target genes. Because Irf4 and Foxo1 expression was found to be down-regulated in vivo and in vitro in Fra-2–deficient B cells, we first analyzed the potential binding of Fra-2 and other AP-1 members on Irf4 and Foxo1 promoters (Fig. 4 c). Indeed, Fra-1, Fra-2, and Jun proteins were able to bind the Irf4 promoter on a TRE element located at −1,986 bp but not −3,429 bp (Fig. 4 c and not depicted). Moreover, ChIP analyses of the AP-1 members in Fra-2–deficient cells revealed that c-Jun, JunD, and JunB are the potential dimerization partners of Fra-2 on the Irf4 promoter and that Fra-1 cannot compensate for the loss of Fra-2 (Fig. 4 c). Furthermore, we could also show that Fra-2 binds the Ikaros promoter on the known Irf4 sites −235 bp (Fig. 4 d). In accordance, Fra-2 and Irf4 binding on the Ikaros promoter was reduced in Fra-2–deficient cells compared with littermate control (Fig. 4 d), suggesting a possible physical interaction between Fra-2 and Irf4. The consensus TRE elements of Fra-2–binding motifs in B cells revealed that Irf4 might be one of its cofactors (Fig. S3 a). Next, we tested the Irf4-Fra-2 interaction by coimmunoprecipitation in littermate control and Fra-2–deficient B cells. In fact, Irf4 interacts with Fra-2, as shown by coimmunoprecipitation (Fig. 4 e). In addition, Western blotting analysis showed a reduction of Irf4 levels in Fra-2–deficient cells (Fig. 4 f). All together, these data suggest that an interaction of Fra-2 with Irf4 could be essential for Irf4 action on the Ikaros promoter.

Next, we determined whether similar results could be obtained with the Foxo1 promoter. ChIP analyses showed that AP-1 proteins bound to the Foxo1 promoter at the −1,270-bp site and that cJun, JunB, and JunD were the potential dimerization partners of Fra-2 on the Foxo1 promoter (Fig. 4, g and h). Moreover, in the absence of Fra-2, Foxo1 showed a reduction of its binding on the promoter of its target genes IL-7 receptor (Fig. 4 i) and Rag (Fig. 4 j). In addition, Foxo1 protein was also decreased in B cells deficient for Fra-2 (Fig. 4 f). Altogether, these results indicate that Fra-2 transcriptionally regulates Irf4 and Foxo1, which through the regulation of their downstream targets Ikaros, IL7Ra, and Rag 1/2 control B cell development (Fig. 4 k).

Foxo1 and Irf4 overexpression rescued the proliferation defect of Fra-2ΔB cell B cell

To determine whether the reduced levels of Foxo1 or Irf4 proteins were responsible for the decreased B cell numbers in Fra-2–deficient mice and the proliferation defect in response to IL-7, restoration of Irf4 and Foxo1 expression was performed in vitro. Indeed, reexpression of Foxo1 or Irf4 by retroviral transduction in IL-7–stimulated Fra-2ΔB cell pro–B cells (Fig. S4 a) led to full rescue of pre–B cell differentiation (Fig. 5 and Fig. S4 b). As expected, the number of small pre–B cells and immature B cells was lower in Fra-2ΔB cell B cells compared with controls after infection with the control vector (Fig. 5, a and b). However, the frequency of Fra-2ΔB cell small pre–B cells was rescued by retroviral transduction of Foxo1 and Irf4. Although the number of immature B cells in Fra-2ΔB cell B cells was still decreased after reexpression of Foxo1 (Fig. 5, a and b), Fra-2ΔB cell pro–B cells expressing Foxo1 or Irf4 showed rescued proliferation (Fig. 5 c). These data indicate that Fra-2–mediated expression of Foxo1 is required, probably via Irf4, but not sufficient for immature B cell differentiation. Moreover, mRNA expression levels of Ikaros, Aiolos, and Rag2 were fully recovered after Irf4 and Foxo1 overexpression, while the expression of Rag1 was still decreased in Fra-2ΔB cell pro–B cells after Foxo1 expression (Fig. 5 d and Fig. S5). The incomplete recovery of Rag1 expression could account for the incomplete rescue of immature B cells. Altogether, these data suggest that Irf4 and Foxo1 are responsible for B cell proliferation and differentiation arrest in Fra-2–deficient cells.

Discussion

In this study, we reveal a hitherto unknown role for the Fra-2/AP-1 transcription factor in early and peripheral B cell development at several hierarchical levels. First, Fra-2 controls proliferation of B220+ B cells in the bone marrow and proliferation of pro–B cells in IL-7–stimulated cultures. Second, Fra-2 controls the differentiation step from large to small pre–B cells. Third, Fra-2 controls the differentiation of immature B cells, and fourth, Fra-2 controls the generation of B2 B cells from T2 precursors in the spleen. Fra-2 exerts its effects through two mechanisms: by binding and thereby controlling the expression of several transcription factors, such as Ebf1, Foxo1, Irf4, and Ikaros, endowed in B cell development and differentiation and by interacting with Irf4 to regulate Irf4 target genes. In accordance with Fra-2–mediated control of expression of Foxo1 and Irf4, the transcription of their target genes Aiolos, Ikaros, Rag 1/2, and IL7Rα is also down-regulated in the absence of Fra-2, thereby explaining the role of Fra-2 in pro–B cell proliferation and pre–B cell differentiation (Fig. 5 e).

B cell development is tightly controlled by the plural action of transcription factors able to guide B cell commitment, survival, proliferation, Ig rearrangement, receptor editing, and final differentiation into plasma cells (Nutt and Kee, 2007; Dengler et al., 2008; Ochiai et al., 2012). Fra-2 down-regulation in B cells leads to reduced numbers of pro, pre, immature, and mature B cells in the bone marrow because of impaired B cell proliferation as measured by BrdU incorporation of pro– and pre–B cells. Within the B cell lineage only pro– and pre–B cells divide in the bone marrow (Carsetti, 2000). The reduced number of B cells in Fra-2–deficient bone marrow was likely due to decreased numbers of pro–B cells in the G1/G0 phase and decreased numbers of pre–B cells in the S phase of the cell cycle. These data strongly imply a function of Fra-2 in B cell proliferation, similar to other members of the AP-1 family previously shown to positively regulate proliferation in other cell types (Shaulian and Karin, 2001; Mercer et al., 2011). The effects exerted by Fra-2 were also directly measured in IL-7–stimulated B cell cultures in vitro. It is possible that deletion of Fra-2 rendered pro/pre–B cells refractory to IL-7, as we showed that Fra-2 also up-regulates Foxo1, which in turn promotes IL7Rα expression (Ochiai et al., 2012). Importantly, we demonstrated that reconstitution of Foxo1 expression in Fra-2–deficient pro/pre–B cells restored responsiveness to IL-7 again. In addition to a direct effect of Fra-2 on IL-7 induced proliferation, we revealed that Fra-2 decreased adhesion of pro–B cells to VCAM-1. In vivo, Fra-2 might therefore be required to support mobilization of pro–B cells toward IL-7 “high” niches during their development (Park et al., 2013). Interestingly, Fra-2 was up-regulated in pro–B cells after IL-7 stimulation by an as yet unidentified pathway. As Fra-2 controls IL7Rα expression and vice versa, up-regulation of Fra-2 by IL-7 constitutes a positive feedback loop supporting the proliferation of pro–B cells because IL-7 is a cytokine essential for lymphocyte development and proliferation (Kittipatarin and Khaled, 2007). In T cells, IL-7 promotes JNK activation and induces the expression of JunD, another member of AP-1 transcription factor (Ruppert et al., 2012), which supports our notion of JunD as partner of Fra-2 on the Irf4 promoter.

A reduction of Fra-2 expression in mature B cells was observed after LPS or CD40L/BCR/IL-4 stimulation, suggesting a specific role of Fra-2 during IL-7 signaling. Indeed, each AP-1 transcription factor might have time- and signal-dependent action in B cells (Ohkubo et al., 2005; Grötsch et al., 2014). As a consequence of the Fra-2/IL-7 loop, we hypothesize that each AP-1 transcription factor has a specific action on B cell physiology by the regulation of a network of target genes. In fact, despite protein similarity between Fra-1 and Fra-2, their action seems to be different in B cells, as increased proliferation was reported in mature B cells lacking Fra-1 (Grötsch et al., 2014). Fra-1 was shown to directly regulate Blimp1 expression at the promoter level in competition with Fos in activated mature B cells. In contrast, loss of Fra-2 affects the expression of genes essential for early B cell development, such as Ebf1, Foxo1, Irf4, Ikaros, Aiolos, and Rag1/2. These data are in accordance with a previous study in which transient expression of cJun and cFos AP-1 transcription factors preceded increased Ebf1 and Foxo1 expression, already suggesting an important role of the AP-1 members in their regulation (Mercer et al., 2011). It was previously shown that Foxo1 is important for both early pro–B cell development and pre–B cell development (Dengler et al., 2008). Moreover, Ikaros is required for the transition of large to small pre–B cells (Lu, 2008; Heizmann et al., 2013; Joshi et al., 2014; Schwickert et al., 2014). Thus, the phenotypes of Fra-2ΔB cell mice resemble those deficient of Foxo1 and Ikaros in the B cell lineage and support our finding that Fra-2 controls Foxo-1 and Ikaros expression. At the molecular level, lack of Foxo1 impaired the expression of IL7Rα and Rag1/2 (Herzog et al., 2009). Intriguingly, we observed decreased Foxo1, IL7Rα, and Rag1/2 expression in Fra-2ΔB cell mice, supporting our notion that regulation of IL7Rα and Rag1/2 expression by Fra-2 is mediated via Foxo-1. Fra-2 seems to be upstream in this hierarchy.

In addition, Fra-2 appears to induce a second transcriptional pathway, as expression of Foxo1 could not fully restore Rag1 expression and immature B cell differentiation. This pathway could be linked to the up-regulation of Irf4. In support of this notion, we have shown that Fra-2 regulates not only Foxo1 but also Irf4, which exhibit important roles during B cell development (Lu, 2003, Lu, 2008). Irf4/8 double-knockout mice show arrested B cell development at the cycling pre–B cell stage, with absence of immature IgM+ B cells and high levels of pre-BCR expression (Lu et al., 2003). Molecularly, both Irf4 and 8 induce germline transcription and promote L chain rearrangement and transcription (Ma et al., 2008). Interestingly, when looking at downstream targets of Foxo1 and Irf4 by ChIP analyses, we showed that (1) Fra-2 together with Jun proteins bound at Foxo1 and Irf4 promoters, (2) Fra-2 together with Irf4 bound to the Ikaros promoter, and (3) Irf4 induced Ikaros and Aiolos expression in pre–B cells dependent of Fra-2. These findings add another level of regulation to the growth inhibition and cell cycle arrest induced by Irf4 or the regulation of pre-BCR and the cell cycle withdrawal mediated by Ikaros, Aiolos, and Irf4, respectively (Ma et al., 2008). Functionally, we could also link the increased adherence of Fra-2ΔB cell B cell to the inactivation of Ikaros, which exhibits aberrant pro–B cells characterized by increased cell adhesion (Schwickert et al., 2014). The impaired down-regulation of the surrogate chain and the decreased expression of κ light chain found in Fra-2ΔB cell mice (unpublished data) could therefore be directly dependent on the positive regulation of Irf4 transcription by Fra-2; however, additional studies are necessary to further characterize this phenotype. In accordance with impaired Ig rearrangement due to decreased Rag1/2 expression, reduced pro–B cell proliferation, and impaired small pre–B cell differentiation, Fra-2ΔB cell mice presented reduced peripheral B cell numbers and concomitantly reduced abundance of serum Igs. The genomic network governed by Fra-2 extends beyond the rather well-characterized factors Foxo1 and Irf4, as shown by our genomic ChIP-seq analysis of the transcriptional Fra-2 network in B cells. The positive regulation of Irf4 by Fra-2 can also explain the differentiation defect of marginal-zone B cells in Fra-2ΔB cell mice, as impaired marginal-zone B cell differentiation is also seen in B cells from Irf4 knockout mice.

In summary, we demonstrate that Fra-2 acts as a key enhancer of B cell proliferation and differentiation. Fra-2 regulates B cell proliferation by specifically binding to Foxo1 and Irf4 promoters, inducing their expression. Fra-2 regulation of Foxo1 and Irf4 is essential for the downstream activation of Ikaros, Aiolos, and Rag1/2 expression, thereby regulating early B cell proliferation and differentiation.

Materials and methods

Animals

To generate B cell–specific Fra-2 deleted mice, Fra-2 floxed mice (Eferl et al., 2007) were crossed with Mb1-cre mice (129S-C57BL/6 mixed background; Hobeika et al., 2006). All mice were maintained on a mixed C57BL/6 background and in a specific pathogen–free facility. Littermate mice (Fra-2 +/+ Mb1-cre tg and Fra-2 floxed Mb1-Cre-) were used as controls in this study. 6-wk-old male and female mice were used in all experiments. Genotyping primers were as follows: Fra-2 lox forward: 5′-GAGGGAGTTGGGGATAGAGTGGTA-3′; Fra-2 lox reverse: 5′-GGACAGCAGGTCAGGAGTAGATGA-3′; Mb1Cre forward: 5′-ACCTCTGATGAAGTCAGGAAGAAC-3′; and Cre reverse: 5′-GGAGATGTCCTTCACTCTGATTCT-3′. Animal experiments were approved by the local ethics committee of Regierung von Mittelfranken, Germany.

Antibodies, flow cytometry, and cell sorting

The following antibodies were used for flow cytometry: Alexa Fluor 647–conjugated anti-CD19, FITC-conjugated anti-CD21/CD35, phycoerythrin (PE)–conjugated anti-CD23, FITC-conjugated anti-CD45R (B220), allophycocyanin-conjugated anti-CD138, PE-conjugated streptavidin (all from BD), PE-conjugated anti-kappa (Antikörper-online.de), PE-conjugated anti-lambda (Antikörper-online.de), PE-conjugated anti-CD25 (BioLegend), PE-conjugated anti-CD2 (BioLegend), PerCP-conjugated anti-CD45R (B220; Miltenyi Biotec), bioconjugated anti-IgM (eBioscience), anti-CD117 (c-Kit; eBioscience), FITC-conjugated anti-CD2 (eBioscience), FITC-conjugated anti-IgD (SouthernBiotech), and Cy5-conjugated anti-IgM (SouthernBiotech). Analyses of expression of cell surface molecules on a single cell level were performed by flow cytometry with a FACSCalibur (BD) or Gallios (Beckman Coulter) flow cytometer. Cells from bone marrow (1.2 × 106) or spleen (2 × 106) were stained with sufficient amounts of FACS antibodies for 20 min at 4°C in the dark. Afterward, cells were washed with 1 ml FACS buffer (2% FCS and PBS) and suspended in 300 µl FACS buffer or stained with a secondary antibody. For analysis of intracellular proteins, cells were fixed and permeabilized with the Fix&Perm kit (An der Grub) according to the manufacturer’s instructions, stained, and analyzed as mentioned before.

For cell sorting, bone marrow cells or splenocytes were incubated in 1 ml antibody solution in 2% FCS and 2 mM EDTA in PBS buffer (MACS buffer) for 30 min at 4°C and then sorted as follows: pro–B cells B220+/c-Kit+/CD19+, T1 B cells CD23/IgM+/CD21, T2 B cells CD23+/CD21+/IgM+, follicular B cells, IgMlow/CD23+/CD21+; MZ B cells, CD23/IgM+/CD21+. Cells were sorted using a MoFlo cell sorter (Dako). Purities of 98–99% were generally achieved.

Isolation of B cells from bone marrow and spleen

Bone marrow was flushed with PBS. Spleens were digested in 5 ml Hank’s buffer supplemented with 1 mg/ml collagenase D (Roche) and 200 U/ml DNase I (Roche) for 30 min at 37°C. The reaction was stopped with 150 µl 0.5 M EDTA, and the tissue was squashed through a 70-µm cell strainer before single-cell suspension. Cells from both organs were treated during 5 min with erythrocyte lysis buffer and afterward suspended in 1 ml of in RPMI1640 medium (10% FCS, 1 mM pyruvate, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 50 µM β-mercaptoethanol; Gibco).

In vitro stimulation of pro– and splenic B cells

Pro–B cells were sorted on the basis of their surface markers using a MoFlo cell sorter (Dako), and B cells from spleen were purified by positive selection on CD45R/B220 MACS beads (Miltenyi Biotec). Pro–B cells were cultured in OptiMEM (10% FCS, 1 mM pyruvate, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 50 µM β-mercaptoethanol; Gibco) and stimulated with IL-7 (5 ng/ml). Splenocytes were resuspended in 90 µl MACS buffer (2% FCS, 0.25 mM EDTA, and PBS) and 10 µl CD45R/B220 MACS beads (Miltenyi Biotec) per 107 cells. After 20 min of incubation at 4°C, cells were washed with MACS buffer and sorted with LS columns. B220+ B cells from spleen were cultured in RPMI1640 (10% FCS, 1 mM pyruvate, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 50 µM β-mercaptoethanol) and stimulated with 10 µg/ml LPS, 10 µg/ml LPS+100 U/ml recombinant mouse IL-4 (Miltenyi Biotec), 10 µg/ml LPS + anti-BCR antibody b.7.6, anti-CD40 mAb (clone FGK), or a mixture of 10 µg/ml anti-BCR antibody b.7.6 anti-CD40 mAb (clone FGK) and IL-4.

Cell cycle analysis

Mice were injected i.p. with 1 mg BrdU for 40 min. Single-cell suspensions were prepared from bone marrow and were stained for cell surface proteins with anti-B220, anti-cKit, anti-CD25, and anti-CD19. Cultured pro–B cells were treated for 45 min with 10 µM BrdU. BrdU was stained with an APC BrdU Flow kit (557892) according to the manufacturer’s protocol (BD). Surface-stained cells were fixed, permeabilized, and treated with DNase I. Incorporated BrdU was stained with anti-BrdU, and total DNA was stained with 7-amino-actinomycin D. Single cells were analyzed using a Gallios flow cytometer, and data were evaluated using Kaluza software.

Proliferation analysis and cell death

For proliferation analysis, 5 × 106 pro–B cells or B220+ B cells from spleen were mixed 1:1 with 10 µM eFluor-solution in PBS (cell proliferation dye eFluor 670; eBioscience). Cell solution was incubated for 10 min at 37°C, stopped with 4–5 volumes OptiMEM (10% FCS, 1 mM pyruvate, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 50 µM β-mercaptoethanol) or RPMI1640 medium (10% FCS, 1 mM pyruvate, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 50 µM β-mercaptoethanol) and incubated for 5 min at 4°C. The stained cells were resuspended in OptiMEM or R10+ and either measured at day 0 by flow cytometry or at day 3 after stimulation with IL-7, LPS, or CD40/BCR/IL-4. The number of achieved cell divisions was calculated using the proliferation analysis option in FlowJo or with the software from FACSCalibur. For cell death analysis, stimulated cells were stained with FITC–annexin V for 20 min in annexin V binding buffer (140 mM NaCl, 20 mM Hepes, pH 7.2, and 2.5 mM CaCl2), washed, resuspended in the presence of 200 µl PI, and measured by flow cytometry.

Adhesion assay

12-well plates were coated for 1.5 h at 22°C with VCAM-1 (10 µg/ml; 643-VM, R&D Systems) and washed three times with D-PBS, followed by the addition of 105 pro–B cells in 0.5 ml OptiMEM medium containing IL-7 (5 ng/ml) and supplemented with 100 ng/ml CXCL12 (SDF-1α; Sigma-Aldrich). After 6 h of incubation at 37°C, the plates were washed with medium and with D-PBS, followed by fixation for 15 min at 22°C with 4% paraformaldehyde. The plates were washed three times with DPBS and stained for 30 min with 0.1% Crystal Violet and then washed with distilled water and dried. Cells attached to the wells were manually counted as viewed with a microscope. For each genotype, the cells from 12 wells (1.12 cm2) were counted to determine the cells per square centimeter.

RNA isolation and reverse transcription

Total RNA was isolated with the TRIzol or TriFast protocol (Invitrogen and Peqlab Biotechnologie, respectively). cDNA synthesis was performed using 0.50–2 µg RNA with the cDNA Reagents from Applied Biosystems (Thermo Fisher Scientific).

Real-time PCR analyses

Quantitative PCR reactions were performed using SYBR Green (Eurogentec Deutschland). Primers for genes analyses and primers for ChIP analyses used for real-time PCR are available upon request. The comparative CT method was used to quantify the amplified fragments. RNA expression levels were analyzed in duplicate and normalized to a housekeeping gene such as β-actin, whereas chromatin-bound fragments were normalized to the same fragments amplified from input chromatin.

ChIP

ChIP experiments were performed with ChIP-IT Express kit (Active Motif) according to the manufacturer’s protocol. 14 µl FosB, c-Fos, Fra-1, Fra-2, JunB, JunD antibodies (Santa Cruz Biotechnology, Inc.), Irf4 (MUM1; Abcam), cJun, and Foxo1 (Cell Signaling Technology) as well as a rabbit control IgG were used for the immunoprecipitation.

ChIP-seq

DNA was sequenced on an Illumina HiSeq 2500 system, using a single end protocol and 100-bp read length. Reads were aligned to the mm10 mouse reference sequence using bwa version 0.7.8-r455 (Li and Durbin, 2009). Peak calling was performed with MACS version 1.3.7.1 (Zhang et al., 2008) for each of the three Fra2 samples against all three controls. Overlapping peaks from all three Fra2 samples were then used for further analysis. Motif discovery was performed with HOMER version 4.7.2 (Heinz et al., 2010) and a size parameter of 200 bp.

Transient transfection of adherent cells using calcium phosphate

Phoenix-eco cells (4 × 106) were seeded in a 10-cm well plate, 1 d before transfection, and incubated overnight at 37°C and 5% CO2 in D10 medium. 20 µg DNA was mixed with 125 µl (2 M CaCl2) and 50 µl (10 mM) chloroquine to a final volume of 1 ml with distilled water. Under bubbling conditions, the mixture was added to 1 ml 2× HBS (50 mM Hepes, 10 mM potassium chloride, 12 mM dextrose, 280 mM sodium chloride, and 1.5 mM Na2HPO4, pH 7.4). Precipitates were trickled onto cells, and after 6–8 h of incubation at 37°C and 5% CO2, cells were provided with 6 ml of fresh D10 medium. Viral supernatant was removed after 24, 48, and 72 h, filtered, and stored at −80°C for infection.

Infection of primary stimulated B lymphoid cells

Pro–B cells (0.5 × 106) were stimulated for 24 h with IL-7 (5 ng/ml) and then infected with retroviral supernatants supplemented with 2 µl transfection reagent (QIAGEN). Infection mixture was centrifuged for 3.5 h at 33,000 rpm at 33°C. Infected cells were stimulated for 3 d with IL-7 and analyzed by flow cytometry.

Coimmunoprecipitation assay

Dynabeads Protein G (Thermo Fisher Scientific) was incubated with 50 µg Fra-2 (Santa Cruz Biotechnology, Inc.) for 10 min at 22°C. Afterward, immunoprecipitation assay was performed with proteins from bone marrow B220+ B cells according to the manufacturer’s protocol. In brief, 100 µl of protein solution was incubated for 30 min with the complex Dynabeads Protein G-Fra-2 antibody. The target antigen was eluted under nondenaturing conditions, added 20 ml Elution Buffer (Thermo Fisher Scientific), and incubated with rotation for 2 min at 22°C to dissociate the complex. The eluted protein was stored at −80°C until Western blot analysis.

Western blot

Proteins from bone marrow B220+ B cells or from immunoprecipitation assay were electrophoresed on 10% SDS polyacrylamide gels and transferred to a nitrocellulose membrane (1.5 h, 110 mA). The efficiency of the transfer was determined with Ponceau S solution. Membranes were blocked in 5% milk powder in TBS-T and probed with anti-Irf4 (MUM1; Abcam), anti-Foxo1 (Cell Signaling Technology), and anti–β actin (Sigma-Aldrich) antibodies at 4°C overnight. Membranes were washed in TBS-T (3 × 15 min) and incubated with the secondary antibody anti–mouse/rabbit IgG HRP conjugate (1:20,000; Promega) for 1 h at room temperature. Bands were detected by ECL (Thermo Fisher Scientific).

Statistical analysis

All experiments were repeated at least three times and done in triplicate. All statistical analyses were performed using Prism (GraphPad Software). Statistical analysis was performed using Student’s t test, and P < 0.05 was accepted as significant. Data are shown as means, and error bars represent standard deviations.

Online supplemental material

Fig. S1 depicts expression of Fra-2 in B cell lineages. Fig. S2 depicts in vitro IL-7 stimulations of B cells from Fra-2ΔB cell and littermate control mice. Fig. S3 shows the Fra-2 recognition sequence in B220+ bone marrow B cells. Fig. S4 shows in vitro LPS or CD40/BCR/IL-4 stimulations of Fra-2ΔB cell and littermate control B cells. Fig. S5 depicts rescue expression of B cell genes.

Acknowledgments

We thank Dr. Michael Reth and Dr. Elias Hobeika for providing the Mb1 Cre mice.

The project was supported by Deutsche Forschungsgemeinschaft (CRC1181, SPP1468, IMMUNOBONE; BO3811/1-1, Emmy Noether, WE 4892/3-1) FP7 Osteoimmune.

The authors declare no competing financial interests.

References

References
Amin
,
R.H.
, and
M.S.
Schlissel
.
2008
.
Foxo1 directly regulates the transcription of recombination-activating genes during B cell development
.
Nat. Immunol.
9
:
613
622
.
Bozec
,
A.
,
L.
Bakiri
,
M.
Jimenez
,
T.
Schinke
,
M.
Amling
, and
E.F.
Wagner
.
2010
.
Fra-2/AP-1 controls bone formation by regulating osteoblast differentiation and collagen production
.
J. Cell Biol.
190
:
1093
1106
.
Carsetti
,
R.
2000
.
The development of B cells in the bone marrow is controlled by the balance between cell-autonomous mechanisms and signals from the microenvironment
.
J. Exp. Med.
191
:
5
8
.
Clark
,
M.R.
,
M.
Mandal
,
K.
Ochiai
, and
H.
Singh
.
2014
.
Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling
.
Nat. Rev. Immunol.
14
:
69
80
.
Dengler
,
H.S.
,
G.V.
Baracho
,
S.A.
Omori
,
S.
Bruckner
,
K.C.
Arden
,
D.H.
Castrillon
,
R.A.
DePinho
, and
R.C.
Rickert
.
2008
.
Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation
.
Nat. Immunol.
9
:
1388
1398
.
Eferl
,
R.
,
A.
Hoebertz
,
A.F.
Schilling
,
M.
Rath
,
F.
Karreth
,
L.
Kenner
,
M.
Amling
, and
E.F.
Wagner
.
2004
.
The Fos-related antigen Fra-1 is an activator of bone matrix formation
.
EMBO J.
23
:
2789
2799
.
Eferl
,
R.
,
R.
Zenz
,
H.-C.
Theussl
, and
E.F.
Wagner
.
2007
.
Simultaneous generation of fra-2 conditional and fra-2 knock-out mice
.
Genesis.
45
:
447
451
.
Grötsch
,
B.
,
S.
Brachs
,
C.
Lang
,
J.
Luther
,
A.
Derer
,
U.
Schlötzer-Schrehardt
,
A.
Bozec
,
S.
Fillatreau
,
I.
Berberich
,
E.
Hobeika
, et al
2014
.
The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells
.
J. Exp. Med.
211
:
2199
2212
.
Heinz
,
S.
,
C.
Benner
,
N.
Spann
,
E.
Bertolino
,
Y.C.
Lin
,
P.
Laslo
,
J.X.
Cheng
,
C.
Murre
,
H.
Singh
, and
C.K.
Glass
.
2010
.
Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities
.
Mol. Cell
.
38
:
576
589
.
Heizmann
,
B.
,
P.
Kastner
, and
S.
Chan
.
2013
.
Ikaros is absolutely required for pre-B cell differentiation by attenuating IL-7 signals
.
J. Exp. Med.
210
:
2823
2832
.
Herzog
,
S.
,
M.
Reth
, and
H.
Jumaa
.
2009
.
Regulation of B-cell proliferation and differentiation by pre-B-cell receptor signalling
.
Nat. Rev. Immunol.
9
:
195
205
.
Hobeika
,
E.
,
S.
Thiemann
,
B.
Storch
,
H.
Jumaa
,
P.J.
Nielsen
,
R.
Pelanda
, and
M.
Reth
.
2006
.
Testing gene function early in the B cell lineage in mb1-cre mice
.
Proc. Natl. Acad. Sci. USA.
103
:
13789
13794
.
Huo
,
L.
, and
T.L.
Rothstein
.
1995
.
Receptor-specific induction of individual AP-1 components in B lymphocytes
.
J. Immunol.
154
:
3300
3309
.
Huo
,
L.
, and
T.L.
Rothstein
.
1996
.
Isolation and characterization of murine fra-1: induction mediated by CD40 and surface Ig is protein kinase C dependent
.
J. Immunol.
157
:
3812
3818
.
Joshi
,
I.
,
T.
Yoshida
,
N.
Jena
,
X.
Qi
,
J.
Zhang
,
R.A.
Van Etten
, and
K.
Georgopoulos
.
2014
.
Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia
.
Nat. Immunol.
15
:
294
304
.
Karin
,
M.
,
Z.
Liu
, and
E.
Zandi
.
1997
.
AP-1 function and regulation
.
Curr. Opin. Cell Biol.
9
:
240
246
.
Kittipatarin
,
C.
, and
A.R.
Khaled
.
2007
.
Interlinking interleukin-7
.
Cytokine.
39
:
75
83
.
Lawson
,
V.J.
,
D.
Maurice
,
J.D.
Silk
,
V.
Cerundolo
, and
K.
Weston
.
2009
.
Aberrant selection and function of invariant NKT cells in the absence of AP-1 transcription factor Fra-2
.
J. Immunol.
183
:
2575
2584
.
Li
,
H.
, and
R.
Durbin
.
2009
.
Fast and accurate short read alignment with Burrows-Wheeler transform
.
Bioinformatics
.
25
:
1754
1760
.
Liebermann
,
D.A.
,
B.
Gregory
, and
B.
Hoffman
.
1998
.
AP-1 (Fos/Jun) transcription factors in hematopoietic differentiation and apoptosis
.
Int. J. Oncol.
12
:
685
700
.
Lin
,
Y.C.
,
S.
Jhunjhunwala
,
C.
Benner
,
S.
Heinz
,
E.
Welinder
,
R.
Mansson
,
M.
Sigvardsson
,
J.
Hagman
,
C.A.
Espinoza
,
J.
Dutkowski
, et al
2010
.
A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate
.
Nat. Immunol.
11
:
635
643
.
Lu
,
R.
2008
.
Interferon regulatory factor 4 and 8 in B-cell development
.
Trends Immunol.
29
:
487
492
.
Lu
,
R.
,
K.L.
Medina
,
D.W.
Lancki
, and
H.
Singh
.
2003
.
IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development
.
Genes Dev.
17
:
1703
1708
.
Ma
,
S.
,
S.
Pathak
,
L.
Trinh
, and
R.
Lu
.
2008
.
Interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to down-regulate pre-B-cell receptor and promote cell-cycle withdrawal in pre-B-cell development
.
Blood.
111
:
1396
1403
.
Marshall
,
A.J.
,
H.E.
Fleming
,
G.E.
Wu
, and
C.J.
Paige
.
1998
.
Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression
.
J. Immunol.
161
:
6038
6045
.
Melchers
,
F.
1995
.
B cell differentiation in bone marrow
.
Clin. Immunol. Immunopathol.
76
:
S188
S191
.
Mercer
,
E.M.
,
Y.C.
Lin
,
C.
Benner
,
S.
Jhunjhunwala
,
J.
Dutkowski
,
M.
Flores
,
M.
Sigvardsson
,
T.
Ideker
,
C.K.
Glass
, and
C.
Murre
.
2011
.
Multilineage priming of enhancer repertoires precedes commitment to the B and myeloid cell lineages in hematopoietic progenitors
.
Immunity.
35
:
413
425
.
Milne
,
C.D.
, and
C.J.
Paige
.
2006
.
IL-7: a key regulator of B lymphopoiesis
.
Semin. Immunol.
18
:
20
30
.
Milne
,
C.D.
,
H.E.
Fleming
, and
C.J.
Paige
.
2004
.
IL-7 does not prevent pro-B/pre-B cell maturation to the immature/sIgM(+) stage
.
Eur. J. Immunol.
34
:
2647
2655
.
Nutt
,
S.L.
, and
B.L.
Kee
.
2007
.
The transcriptional regulation of B cell lineage commitment
.
Immunity.
26
:
715
725
.
Ochiai
,
K.
,
M.
Maienschein-Cline
,
M.
Mandal
,
J.R.
Triggs
,
E.
Bertolino
,
R.
Sciammas
,
A.R.
Dinner
,
M.R.
Clark
, and
H.
Singh
.
2012
.
A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation
.
Nat. Immunol.
13
:
300
307
.
Ohkubo
,
Y.
,
M.
Arima
,
E.
Arguni
,
S.
Okada
,
K.
Yamashita
,
S.
Asari
,
S.
Obata
,
A.
Sakamoto
,
M.
Hatano
,
J.
O-Wang
, et al
2005
.
A role for c-fos/activator protein 1 in B lymphocyte terminal differentiation
.
J. Immunol.
174
:
7703
7710
.
Park
,
S.-Y.
,
P.
Wolfram
,
K.
Canty
,
B.
Harley
,
C.
Nombela-Arrieta
,
G.
Pivarnik
,
J.
Manis
,
H.E.
Beggs
, and
L.E.
Silberstein
.
2013
.
Focal adhesion kinase regulates the localization and retention of pro-B cells in bone marrow microenvironments
.
J. Immunol.
190
:
1094
1102
.
Ruppert
,
S.M.
,
M.
Chehtane
,
G.
Zhang
,
H.
Hu
,
X.
Li
, and
A.R.
Khaled
.
2012
.
JunD/AP-1-mediated gene expression promotes lymphocyte growth dependent on interleukin-7 signal transduction
.
PLoS One.
7
:
e32262
.
Schwickert
,
T.A.
,
H.
Tagoh
,
S.
Gültekin
,
A.
Dakic
,
E.
Axelsson
,
M.
Minnich
,
A.
Ebert
,
B.
Werner
,
M.
Roth
,
L.
Cimmino
, et al
2014
.
Stage-specific control of early B cell development by the transcription factor Ikaros
.
Nat. Immunol.
15
:
283
293
.
Shaulian
,
E.
, and
M.
Karin
.
2001
.
AP-1 in cell proliferation and survival
.
Oncogene.
20
:
2390
2400
.
Tilzey
,
J.F.
,
T.C.
Chiles
, and
T.L.
Rothstein
.
1991
.
Jun-B gene expression mediated by the surface immunoglobulin receptor of primary B lymphocytes
.
Biochem. Biophys. Res. Commun.
175
:
77
83
.
Zhang
,
Y.
,
T.
Liu
,
C.A.
Meyer
,
J.
Eeckhoute
,
D.S.
Johnson
,
B.E.
Bernstein
,
C.
Nusbaum
,
R.M.
Myers
,
M.
Brown
,
W.
Li
, and
X.S.
Liu
.
2008
.
Model-based analysis of ChIP-Seq (MACS)
.
Genome Biol.
9
:
R137
.

    Abbreviations used:
     
  • BCR

    B cell receptor

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • ChIP-seq

    ChIP sequencing

  •  
  • Ebf

    early B cell factor

  •  
  • Foxo

    forkhead box protein

  •  
  • Irf

    interferon regulatory factor

  •  
  • VCAM

    vascular cell adhesion protein

Author notes

*

D. Mielenz and A. Bozec contributed equally to this paper.

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Supplementary data