A microarray screen for Oct2-dependent genes identified the tyrosine kinase Syk as a potential target gene. Characterization of the murine Syk promoter using 5′ RACE identified two alternative transcriptional initiation sites (Figure 1B) that append alternative 5′ non-coding exons to Syk mRNAs in B cells. Both transcripts encode the same protein, as the start of Syk translation lies in an exon common to the two transcripts. RNAseq data for Syk in sorted B cell populations show that the usage of exons 1 or 2 varies subtly among them (Figure 1C).

The promoter upstream of exon 2 is positively regulated by Oct2. B cells sorted from Oct2^−/− mice (Figure 2A) have a lower level of Syk transcripts and protein than wild type (WT) mice (Figures 2B,C). Using qPCR to distinguish Syk transcripts derived from exon 1 or exon 2, we found that those derived from exon 2 were selectively reduced in Oct2^−/− B cells (Figure 2D). To confirm the influence of Oct2 on the Syk exon 2 promoter, we stably introduced an estradiol-inducible form of Oct2 (29) into a cloned Oct2^−/− B lymphoma cell line, OM1 (48). Upon treatment with estradiol, there was no effect on expression from exon 1 with either the vector only control or the inducible Oct2 construct. However, estradiol induction of Oct2 selectively enhanced Syk expression from exon 2 and culminated in markedly increased Syk mRNA levels (Figures 2E,F). A DNA sequence search revealed three perfect consensus octamer sequences in the Syk gene (Figure 1B). Chromatin immunoprecipitation (ChIP) of Oct2 on DNA from the WT B lymphoma WEHI231 showed strong enrichment of DNA from the promoter of the known Oct2 target gene Cd36 (Figure 2G). The DNA adjacent to the perfect octamer sequence upstream of exon 2 was also significantly enriched in the ChIP, but adjacent sequences in intron 1/2 were not. Thus, Oct2 can directly increase Syk levels in B cells, acting at one of two alternative promoters.

As mentioned above, Oct2 is required for the full functional and phenotypic maturation of B cells, such that peripheral B cells in Oct2^−/− mice are numerically reduced and display some features of immature transitional cells, and the B1 and MZ populations are missing (27, 29, 38). Like transitional B cells, Oct2-deficient B cells are killed rather that activated by BCR cross-linking [Ref. (38, 49); see also Figure 3A]. However, Oct2^−/− B cells respond normally to the survival factor Baff/BlyS, and survive to expand normally when BCR signaling occurs in the presence of Baff (Figures 3A,B and data not shown). Oct2^−/− mice expressing a Bcl2 transgene still lack B1 and MZ B cells (data not shown). Thus abnormal survival properties are not responsible for the lack of these two populations in the Oct2 mutant mice.

We speculated that the reduced Syk levels in Oct2^−/− B cells might contribute to their failure to mature in vivo and respond to BCR signals in vitro. We constructed a retroviral vector expressing Syk, and infected and cloned WT (BC1) and Oct2-deficient (OM1) lymphoma cells (Figure 3C). Boosting Syk protein levels enhanced the BCR response, as measured by calcium flux, in both WT and Oct2^−/− cells (Figure 3D). Finally, using transduction of primary B cells (see Materials and Methods), we found that elevating Syk levels improved the proliferation of WT B cells, both unstimulated, and more strongly, upon BCR cross-linking, and that Oct2^−/− B cells complemented with Syk retrovirus were activated to expand, rather than be killed by a BCR signal (Figure 3E). The rescue was not complete, as cell survival was still lower overall in the mutant cell cultures. This is likely to reflect technical limitations of the assay, including comparative infectivity of WT and mutant cells, and the correct timing of exogenous Syk expression in the context of the BCR signal. However, the results strongly suggest that Syk levels are limiting in Oct2^−/− and, to a lesser extent, in WT B cells. We propose that Oct2 regulates Syk gene expression to enable positive selection through the BCR and therefore entrance to the mature follicular B cell pool, and it may similarly enable differentiation of B1 and MZ B cells, which are highly dependent on BCR signal strength.

Slamf1 encodes CD150, a lymphocyte signaling and adhesion molecule (50) that is expressed in B cells and T cells and at very low levels in myeloid cells (see www.immgen.org). Our RNAseq analysis of activated B cells indicated that Slamf1 was expressed at abnormally low levels in Oct2 KO B compared to controls (Figure 4A). This was confirmed by flow cytometric analysis (Figure 4B). Resting WT and LPS-activated B cells expressed similar levels of Slamf1, but levels on Oct2-deficient B cells were much lower than controls under both conditions. Activated WT T cells up-regulated Slamf1 from resting levels. However, Slamf1 was not Oct2-dependent in resting or activated T cells, or in macrophages expanded from fetal liver, despite Oct2 normally being expressed in these cell types (15, 16, 18).

We next asked whether Slamf1 was a direct Oct2 target using cloned WT (WEHI231) and KO (OM1) B lymphoma lines transduced with the inducible Oct2-ER vector, as described above. As expected, the Oct2 target Cd36 gene was expressed in WEHI231 but not OM1 (Figure 4C). Estradiol treatment enhanced Cd36 levels in WEHI231, and strongly induced Cd36 expression in OM1 cells. However, Slamf1 expression, while low in the Oct2^−/− line, was not increased by Oct2 induction. Therefore, Oct2 is unlikely to directly regulate Slamf1 transcription in B cells.

Examination of the pattern of expression of the Slamf1 gene during B cell development, using the Immgen database (http://www.immgen.org) and by flow cytometry of bone marrow (BM) and peripheral B cell populations indicated that Slamf1 is a marker of B cell maturation, appearing during the transition from immature (IgM^hi/IgD^lo) to mature (IgM^lo/IgD^hi) follicular B cells of the spleen (Figure 4D), with MZ B cells expressing intermediate Slamf1 levels. We conclude that loss of Oct2 blocks B cell maturation before the Slamf1⁺ stage, thereby indirectly regulating its expression.

It has been shown that B cells lacking Slamf1 cannot form the lasting interactions with Tfh cells that are required for GC formation (51), and yet Oct2 mice do form GC upon infection and immunization (11). We immunized WT and KO mice with SRBC and stained GC cells for Slamf1 9 days later. Oct2^+/+ and Oct2^−/−GC B cells expressed similar levels of Slamf1. This indicates that Oct2 is dispensable for Slamf1 expression, and that endogenous signals driving the GC response override the Oct2 maturation defect in vivo (Figure 4E). To explore the nature of these signals, we tested the capacity of a number of mitogens and cytokines to induce Slamf1 expression on Oct2-null B cells in vitro. Anti-μ, anti-CD40, Baff, and IL4, tested singly or in all possible combinations, failed to induce appreciable Slamf1 on Oct2^−/− B cells, suggesting that other factors and cells contribute in vivo. Collectively these studies point to an Oct2-regulated differentiation step that enables efficient FoB cell maturation.

We have shown that both Oct2 and Obf1 affect a B cell’s capacity to differentiate to ASC in response to particular cytokines, with Oct2 regulating the response to IL5 (29), and Obf1 being essential for ASC differentiation driven by IL4 (52).

We determined that Obf1 lies downstream of Stat6 in the IL4 signaling cascade (Figures 5A–C). Interestingly, while the IL4/Stat6 axis drives both isotype switching and ASC differentiation (45), Obf1 is dispensable for IL4 driven switching (52). In order to learn how IL4 and Stat6 regulate Obf1 expression, we performed microarray analysis on the WEHI231 B lymphoma, an IL4 responsive line. Because Stat6 exists in a latent form in the cytoplasm, and is activated by phosphorylation to enter the nucleus and act as a transcription factor (53), direct Stat6 targets would be activated after IL4 signaling even in the absence of new protein synthesis. Cells therefore were treated with cyclohexamide (CHX) to inhibit translation, and 1 h later, IL4 was added. A parallel culture did not receive IL4. After another 4 h, RNA was prepared from both. Analysis using Illumina Sentrix Mouse v1.1 arrays identified a small number of genes whose expression rose two to fourfold during this short period of IL4 stimulation, genes likely to be regulated directly by Stat6. Among these were three transcriptions factors: Nfil3, Vdr, encoding the 1,25-dihydroxyvitamin D3 receptor, and Xbp1. Their induction under these conditions was validated by qPCR (Figure 5D). Interestingly, Obf1 transcription was not elevated by IL4 treatment in the presence of CHX, indicating that it is not a direct Stat6 target gene (Figure 5D).

As Obf1 RNA is elevated by IL4 treatment in B cells in the absence of CHX (Figure 5B), we hypothesized that one of the three transcription factors directly regulated by Stat6 might drive Obf1 expression. We therefore constructed ER fusion vectors for each to determine their effects on Obf1 expression. The Xbp1-ER expression vector contained the mature, processed, and active form of this factor (54). Clones of each line were cultured for 24 h unstimulated, with estradiol to induce each fusion protein, or with IL4. IL4 caused an increase in Obf1 RNA levels in all cases, as expected. However, estradiol induction only increased Obf1 levels significantly in the Xbp-ER expressing cell line (Figure 5E). Finally, primary splenic B cells from Xbp1^+/+, heterozygous and conditional KO mice were stimulated for 48 h with CD40 ligand and IL4, and protein extracts prepared. Western blots showed that Xbp1-null B cells express markedly less Obf1 than the controls (Figure 5F). These data indicate that Nfil3 and Vdr do not influence Obf1 expression, but that Xbp1 has the capacity to directly activate the Obf1 gene in response to IL4/Stat6 signaling in B cells.

B lymphoma cell lines used here were all generated in house: WEHI231 (75), BC1, and OM1 (48). Primary splenic B and T cells were purified using anti-B220 or anti-CD4 microbeads (Miltenyi), as described (11). Oct2^+/+ and Oct2^−/−macrophages were expanded in vitro from E13 fetal liver using M-CSF, as described (15). Retroviral transduction in all cases used the pMX-pie vector and was preformed using spin infection as described (48, 76). For lymphoma lines, cells were cloned post-infection as single GFP⁺ cells in puromycin-supplemented medium.

For the retroviral complementation experiment of Figure 3E, primary splenic B220⁺ cells were stimulated for 24 h with CpG [1 μM oligonucleotide CpG 1668 (sequence 5′-TCCATGA CGTTCCTGATGCT-3′), fully phosphothioated GeneWorks] to promote cell cycling and enable retroviral infection. After overnight culture, cells were washed and resuspended in medium at 0.5 × 10⁶ cells/ml, without CpG but containing anti-μ [10 μg/ml AffiniPure F(ab′)₂ fragment, goat anti mouse Jackson Laboratories] and/or Baff (250 ng/ml a kind gift from Jürg Tschopp). Cell survival in transduced (GFP⁺) cells was assessed after a further 48 h by flow cytometry, propidium iodide exclusion, and cell counting using internal microbead controls.

Retroviruses expressing transcription factors fused to the human estrogen receptor (hER) dimerization domain were generated by amplification of each factor’s ORF, and sequencing each amplified product to ensure it was mutation-free and in frame with the hER. Production of ER fusion proteins of the correct size was confirmed by western blots of infected or transfected cells. Anti-CD40 (clone FGK4.5) was prepared in house and used at 10 μg/ml. β-Estradiol (Sigma) was used at 10 μM, cycloheximide at 50 μM.

Oct2^−/−, Obf1^−/−, and Stat6^−/− mice have been described previously (19, 36) Xbp1^fl/flCd19^Cre/+mice, where Xbp1 is conditionally deleted in the B cell lineage, were generated by Hetz et al. (77) and further described in Taubenheim et al. (72). All mice were maintained on a C57BL/6 background, and all experiments conformed to the relevant regulatory standards of the Animal Ethics Committee of the Walter and Eliza Hall Institute of Medical Research (AEC Projects 2010.010 and 2013.014).

For westerns, antibodies used were specific for Oct2 (clone 9A2, in house), Obf1 (clone 6F10, in house) Stat6 (S-20), Syk (N-19), and actin (I-19) all from Santa Cruz and Xbp1 (Ab37152-100, Abcam). For flow cytometry, antibodies used were specific for B220 (RA3-6B2), IgD (11-26c.2a), fas/CD95 (Jo2), all from BD Pharmingen Slamf1/CD150 (TC15-12 F12.2, Biolegend), and GL7 (eBioscience). Calcium flux was assessed cytometrically as described (78).

Illumina Sentrix Mouse v1.1 arrays were probed with RNA prepared from independent B lymphoma cell lines: to identify Oct2-dependent genes, two independent clones of OM1 cells stably transduced with a control vector or one expressing an estradiol-inducible Oct-ER fusion protein were treated in vitro for 6 or 48 h with estradiol prior to RNA preparation. For Obf1 targets, two clones of the Obf1^−/− BM1 lymphoma line (78), transduced with vector control or an Obf1-ER expression vector, were induced in a similar manner and RNA prepared and analyzed.

Peripheral B cell populations were sorted from naïve or immunized C57Bl/6 mice as described in the legend to Figure 1. Two independent biological replicates were prepared for each population, except for spleen and BM ASC. Because of the paucity of ASC in these tissues, cells were pooled from three to four individuals before sorting. Two such pools were processed independently for sequencing. Normalized expression levels are shown in the graphs. As more than 70% of reads in RNA from ASC map to the Ig loci, all Ig reads were excluded from the data, for all populations, before normalization to generate the values shown in Figure 1A. Specifically, all reads from the IgH locus on chromosome 12, NC_000078.6 (positions 113,258,768–116,009,954), all reads from the Igλ locus on chromosome 16, NC_000082.6 (positions 19,026,858–19,260,844), and all reads from the Igκ locus on chromosome 6, NC_000072.6 (positions 67,555,636–70,726,754) were excluded. For Figure 2B, Oct2^+/+ and Oct2^−/−B cells were sorted, as in Figure 2A, from spleen of two independent mice of each genotype and activated for 48 h before RNA extraction and preparation for sequencing. As these conditions do not induce ASC differentiation, Ig sequence reads were not excluded from analysis of these samples.

The starts of Syk transcription in primary B cells were determined directly using FirstChoice^® RLM-RACE (Ambion) and nested Syk gene specific primers specific for the first Syk coding exon: 5′-GTAGGTCAGGTGGTTGGCGCTGTCCACAGC-3′ and 5′-CCCGCCATGTCTGCACCCCTTCAGAGTTC-3′.

For exon-specific Syk qPCR, these primers were used:

5′Syk exon 1: 5′-CAGTGACTGCGGCTGAGCGCGGACC-3′

For Syk ChIP PCR, these primers were used:

Exon 2: fwd 5′-GCCTAGGCCACGATGGTCAAAGGAGG-3′ and

Chromatin immunoprecipitation was conducted as previously described (29), using high titer, polyclonal anti-Oct2 rabbit serum generated in house.

Primers for other qPCR were:

Stat6: fwd 5′-CTGCTGGGCCGAGGCTTCACATTT-3′

Chromatin immunoprecipitation PCR and qPCR were performed on triplicate samples in all cases, and the data presented as means ± SD. Statistical significance was determined using the unpaired Student’s t-test.

Mice stably reconstituted with Oct2^+/+ or Oct2^−/−fetal liver were immunized i.p with 2 × 10⁹ sheep red blood cells (Applied Biological Product Management, Australia) in 100 μl of PBS and sacrificed after 9 days.