Research Article

Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes

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PNAS March 25, 2014 111 (12) 4513-4518; https://doi.org/10.1073/pnas.1402739111

  1. Contributed by Christopher C. Goodnow, February 13, 2014 (sent for review January 14, 2014)

Significance

Mammalian B lymphocytes make antibodies of five different heavy chain isotypes, IgM, IgD, IgG, IgE, and IgA. The different isotypes are produced at discrete stages in B-cell development from a single immunoglobulin heavy chain (Igh) gene, either by irreversible rearrangement of the gene to make IgG, IgE, or IgA or by alternative splicing of the RNA transcribed from the Igh gene to coexpress IgM and IgD. Developmentally regulated trans-acting factors have been hypothesized to control IgM and IgD expression from large Igh RNAs, but these factors have remained elusive for several decades. Here, using a genome-wide mutation screen in mice, we identify an obscure gene, Zfp318, as encoding a specific and essential factor promoting IgD expression in mature B cells.

Abstract

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.

Ig isotypes with different heavy (H)-chain constant regions are made by B lymphocytes in a developmentally regulated series (1). The different antibody isotypes serve as cell surface markers of B-cell maturation, as functionally distinct receptors for B-cell activation by antigens and as secreted mediators of different antibody effector functions (2). All B cells begin as immature B cells in bone marrow or fetal liver that express only the IgM isotype on their cell surface (3), comprised of H chains with an N-terminal variable domain and C-terminal constant region domains, transmembrane segment, and cytoplasmic tail, paired with Ig light chains. Maturation into follicular B cells, which recirculate among the spleen, lymph nodes, and other secondary lymphoid tissues, is marked by coexpression of a second isotype, IgD. Each mature follicular B cells displays a mixture of cell surface B-cell receptors (BCRs) comprising the same variable domain joined to either IgD or IgM constant regions, with greater levels of IgD than IgM (4, 5). B cells undergo isotype switching after activation by microbial antigens and helper T cells: They irreversibly lose IgM and IgD and switch to expressing the same variable domain linked to IgG, IgA, or IgE constant region domains. Although the process of isotype switching to IgG, IgA, and IgE is well understood, the mechanism for developmentally regulated IgD expression remains obscure.

The developmental order of antibody isotype expression is reflected in the layout of the Ig heavy chain locus, Igh. In surface IgM+ immature B cells, transcription begins with two variable exons (LH and VDJH) formed by intrachromosomal recombination of separate LHV, D, and J elements in pre-B cells. Downstream from the VDJH exon are six Ighm constant region exons encoding the extracellular and transmembrane segments of membrane IgM, then five Ighd constant region exons encoding the corresponding segments of IgD, and finally similar sets of Ighg, Ighe, or Igha exons encoding the constant regions of IgG, IgE, and IgA. Isotype switching results from further DNA recombination within the locus that deletes the Ighm and Ighd exons and brings either the Ighg, Ighe, or Igha exons immediately 3′ to the VDJH exon, so that the latter is spliced to IgG, IgE, or IgA constant region exons in the resulting mRNA (6⇓–8). IgD is the exception, however, because most B cells do not express IgD by DNA recombination but instead via a reversible, developmentally regulated process of alternative mRNA splicing of the VDJH exon to the Ighm and Ighd exons (5, 9, 10). This unique arrangement for coexpression of IgM and IgD mRNA by alternative splicing is conserved in bony fish, amphibians, reptiles, monotremes, and mammals (11), yet it is not known how IgD mRNA is selectively produced in mature B cells.

Pre-B cells and immature B cells express very little IgD mRNA and express only IgM, despite transcribing the Ighd exons at levels that are often only two- to threefold lower than the Ighm exons and not differing between IgD+ and IgD− IgM+ B cells, when measured by RNA-polymerase run-on experiments in isolated nuclei (12⇓⇓⇓–16). These results have led to the hypothesis that 25-kb-long Igh pre-mRNA transcripts traverse from the VDJH exon through the Ighd exons in both immature and mature B cells, but an unknown transacting factor alters splicing either by: (i) promoting RNA cleavage at Ighm polyadenylation sites in immature B cells to preclude VDJH splicing to Ighd; or (ii) silencing Ighm cleavage and polyadenylation sites in mature B cells to allow splicing to Ighd (13). In some immature B cells, failure to express IgD also appears to reflect unloading of RNA Pol II at an attenuation region 3′ to Ighm and 5′ to Ighd, but when this region is removed, there is still little splicing to IgD (16, 17). In contrast, in terminally differentiated plasma cells, transcription termination occurs upstream of Ighd, resulting in very low expression of Ighd mRNA.

Although differential expression of IgM and IgD was one of the first examples of developmentally regulated alternative mRNA splicing, progress to understand its basis has stalled because it has not been possible to identify the nature of the transacting factors. Here, we use a phenotype-driven genetic screen in mice to identify a gene that fulfils the criteria for encoding the elusive transactivating factor promoting IgD expression.

Results

Identification of a Missense Mutation in Zfp318 Causing Decreased IgD and Increased IgM.

In a peripheral blood screen of mice inheriting ethylnitrosourea (ENU)-induced point mutations, we identified a pedigree with a Mendelian recessive mutation characterized by decreased IgD and increased IgM on mature B cells (Fig. 1 A and B). Homozygotes had one-third as much IgD as wild-type littermates and unrelated controls, whereas heterozygotes had an ∼25% decrease. The frequency of B cells was slightly reduced (Table 1). Exome sequencing of an affected animal followed by genotyping of candidate mutations in a large cohort of siblings and offspring revealed that the low IgD trait was completely correlated with inheritance of a point mutation in the gene encoding zinc-finger protein (ZFP) 318.

Fig. 1.
Fig. 1.

Decreased IgD and increased IgM on circulating B cells from mice with a point mutation in the long isoform of Zfp318. (A) Representative flow cytometry of peripheral blood lymphocytes from a homozygous Zfp318 mutant mouse and a wild-type littermate showing the frequency of CD3+ T cells and CD19+ B cells among lymphocytes (Upper), and the frequency of IgMlow IgDhi mature B cells among CD19+ B cells (Lower). (B) Geometric mean fluorescent intensity (MFI) of IgM and IgD on blood CD19+ B cells in Zfp318 homozygous mutant, heterozygous, and wild-type littermates. (C) Relative abundance of Zfp318 and Ighd mRNA measured in arbitrary units in sorted bone marrow pro-B cells and splenic immature (CD93+ IgMhi) and mature (IgMlo CD93−) B cells. (D) Schematic of the two isoforms of Zfp318 generated by alternative splicing of the numbered exons and location of ENU-induced mutation. (E) Evolutionary conservation of the LRID. Highly conserved residues are in bold and the mutated Ile1347 residue in red. Accession nos.: Homo sapiens (human), XP_005249038.1; Pan troglodytes (chimpanzee), XP_518490.3; Mus musculus (mouse), AAI50731.1; Monodelphis domestica (opossum), XP_001365292.1; Falco cherrug (falcon), XP_005439139.1; Gallus gallus (chicken), XP_419507.4; Chelonia mydas (sea turtle), EMP24462.1; Ophiophagus hannah (king cobra), ETE68519.1; Alligator sinensis (alligator), XP_006034767.1; Xenopus tropicalis (frog), XP_004914880.1; Latimeria chalumnae (coelacanth), XP_006013622.1; Metriaclima zebra (cichlid fish), XP_004575369.1; Danio rerio (zebrafish), XP_002664136.3.

View this table:
Table 1.

Frequency of B-cell subpopulations in bone marrow and spleen in Zfp318 point mutant mice and in the blood of Zfp318−/−

ZFP318 is also called testicular zinc-finger protein (TZF) and has been implicated in transcriptional regulation in testes with genetic deficiency causing infertility in mice (18⇓⇓–21) but has no known function in the immune system. Microarray comparisons of gene expression in B-cell subsets have identified Zfp318 as a member of a set of mRNAs that increases during maturation of immature B cells into mature follicular B cells (22⇓–24). By analyzing flow-sorted B-cell subsets, we confirmed expression of Zfp318 mRNA closely parallels IgD heavy chain (Ighd) mRNA during B-cell development (Fig. 1C). There was very little Zfp318 in pro-B cells in the bone marrow, moderate amounts in immature (CD93+, CD62L−) B cells in the spleen, and high amounts in mature follicular (CD93−, CD62L+) B cells. The close correlation between IgD and Zfp318 expression is reinforced in the IMMGEN dataset (22, 23), which shows highest Zfp318 expression in IgDhigh follicular B cells and T3 transitional B cells, lower Zfp318 in IgDlow marginal zone B cells and T1-T2 transitional B cells, and very low expression in IgD-negative IgM+ immature B cells in the bone marrow.

Two alternatively spliced isoforms of Zfp318 mRNA are annotated in the genome and described in the literature (18) (Fig. 1D). The long isoform (CCDS 28827.2) includes exons 1–10 and encodes a 2,237-aa protein containing two C2H2 zinc-finger domains of the U1 ribonucleoprotein type (25). A 44-aa linker separates the two zinc fingers, similar to the 34- to 44-aa linkers between the dsRNA-binding U1-type zinc fingers in the JAZ and ZFa proteins and unlike the 6- to 8-aa linkers typically present between C2H2 zinc fingers in DNA binding proteins (26). The short isoform (CCDS 28826.2) skips exons 8, 9, and 10 and, consequently, encodes a protein of 1,154 aa lacking the second zinc finger and the polyproline domain. The IgD-lowering mutation was a nonsynonymous T > C transition in the differentially spliced exon 10. Because this mutation only alters the mRNA and protein sequence of the long form, it demonstrates that the long Zfp318 isoform promotes normal IgD expression. The mutation changed codon 1347 in the long isoform from a highly conserved hydrophobic isoleucine into a polar threonine. Ile1347 lies in an unannotated domain between the second zinc finger and the polyproline domain, containing 16 lysine residues that are highly conserved in mammals, birds, reptiles, and bony fish (Fig. 1E). We refer to this domain as the lysine-rich IgD-promoting domain (LRID). It is notable that the conserved Ile residue is substituted to Glu in Maylandia zebra and other Cichlid fish species, although we are unaware of any data on IgD expression in these species.

Effect of Zfp318I1347 Mutation on B-Cell Developmental Subsets in Bone Marrow and Spleen.

Analysis of B-cell development in the bone marrow of Zfp318 mutant mice and littermate controls showed no significant difference in either percentage of B cells or subset distribution of developing B cells but a small increase in the proportion of mature recirculating B cells (Fig. 2A and Table 1). Interestingly, the subset of CD93+, IgM+ immature B cells in the bone marrow that starts to express IgD at very low levels already showed a reduced expression of IgD compared with either wild-type (P < 0.0001) or heterozygous (P < 0.001) littermate controls without a statistically significant change of IgM expression (Fig. 2B). In contrast, the mature, recirculating B cells in the bone marrow had reduced expression of IgD and increased expression of IgM (Fig. 2B).

Fig. 2.
Fig. 2.

B-cell development in bone marrow of Zfp318 point mutant mice. (A) Flow cytometry of bone marrow lymphocytes from Zfp318 mutant mice (Hom) and wild-type littermate controls (WT). B cells were gated as B220+ cells. Expression of IgM and IgD was analyzed on CD93+ immature and CD93− mature B cells. (B) MFI of IgM and IgD on immature and mature B cells in the bone marrow gated as in A. Statistical analysis by one-way ANOVA followed by Bonferroni post hoc test: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001.

Splenic B cells in homozygous mutants showed a similarly decreased IgD and increased IgM, but normal expression of other mature B-cell surface markers with the exception of slightly increased CD23 (Fig. 3 A–C). In normal B cells, CD93 expression is extinguished, whereas CD62L, CD23, CD21, and BAFF-Receptor are induced when immature B cells in the spleen mature into recirculating follicular B cells, and these developmentally regulated events were comparable in Zfp318I1347T homozygous mutant and wild-type mice. The two main subsets of mature B cells in the spleen, CD23+ CD21med follicular B cells and CD23− CD21hi marginal-zone B cells, were both present in normal frequencies (Fig. 3A and Table 1). The lower level of IgD expressed on wild-type marginal zone B cells was completely extinguished in Zfp318I1347T homozygotes, representing a decrease of ∼10-fold on marginal zone B cells compared with only ∼threefold decreased IgD on follicular B cells (Fig. 3B).

Fig. 3.
Fig. 3.

B-cell subsets in spleen of Zfp318 point mutant mice. (A) Representative flow cytometry of splenic lymphocytes from Zfp318 mutant mice (Hom) and WT littermate controls showing the frequency of TCRβ+ T cells and B220+ B cells among lymphocytes (Left), frequency of CD93+ immature and CD93-CD62L+ mature B cells (Center Left), frequency of CD23+ follicular and CD21+ marginal zone B cells among CD93− mature B cells as well as straining for IgM and IgD on mature CD93− B cells (Center Right and Right). (B) MFI of IgM and IgD on immature, follicular, and marginal zone B cells in the spleen gated as in A. (C) MFI of antibody staining for BAFF-receptor, CD23, CD21, and CD62L on mature follicular B cells (Upper) and marginal zone B cells (Lower) in the spleen. Statistical analysis by one-way ANOVA followed by Bonferroni post hoc test: ns, P > 0.05; *P < 0.05; **P < 0.01; #P < 0.0001.

Loss of IgD Expression in Mice with a Zfp318 Null Mutation.

The above finding that IgD expression was extinguished on marginal zone B cells but persisted at moderate levels on homozygous mutant follicular B cells had two alternative explanations: (i) another gene might also contribute to IgD expression in follicular B cells; or (ii) the Zfp318I1347T point mutation may only partially compromise ZFP318 function. To resolve these alternatives, we analyzed circulating B cells in mice carrying a null Zfp318 allele generated by a targeted insertion in exon 2. Homozygous Zfp318 null mice had a normal percentage of circulating B cells and a normal fraction of these B cells that had matured to the CD93-negative stage (Fig. 4A), but IgD expression was almost completely abolished on immature and mature B cells (Fig. 4 B and C). IgM was increased threefold on mature B cells from homozygous Zfp318 null mice. Thus, ZFP318 is absolutely required for IgD expression and the point mutation only partially inactivates its function.

Fig. 4.
Fig. 4.

Loss of IgD expression on circulating B cells of mice with a Zfp318 null mutation. (A) Representative flow cytometry of peripheral blood lymphocytes from a Zfp318 homozygous null (−/−) and wild-type littermate control (+/+) showing the frequency of CD3+ T cells and CD19+ B cells among lymphocytes (Left), and the frequency of CD93− mature and CD93+ immature B cells among CD19+ lymphocytes (Right). (B) Expression of IgM (Upper) and IgD (Lower) on B cells from Zfp318 homozygous null (−/−, black line) and wild-type (+/+, shaded). As a negative control, staining on T cells from Zfp318−/− mice is also shown (dashed line). (C) MFI of IgM and IgD on CD93+ immature and CD93− mature B cells from Zfp318−/− mice compared with heterozygous and wild-type littermate controls.

Discussion

The findings above identify ZFP318 as a long-sought transacting factor governing differential expression of IgM and IgD isotypes during B-cell maturation. Earlier work demonstrated that 25-kb-long heavy chain pre-mRNAs are transcribed from the VDJH exon through the Ighm and Ighd exons in both immature and mature B cells (12⇓⇓⇓–16). Consequently, it was hypothesized that the alternative splice acceptor sites on the first Ighm and Ighd constant region exons must compete for the single splice donor sequence in the VDJH exon, with unknown transacting factors either suppressing RNA splicing to the Ighd exons in immature B cells or promoting Ighd splicing in mature B cells at the expense of Ighm. The results here support the latter hypothesis: Zfp318 mRNA increases during B-cell maturation in parallel with increasing IgD, and Zfp318 mutations increase IgM and extinguish IgD expression.

Zfp318 appears to be specifically required for balancing IgD and IgM output from Igh, and not for the overall program of B-cell maturation. Zfp318 mutation had no effect on the accumulation of mature B-cell subsets nor did it diminish the expression of CD21, CD62L, or CD23 on mature B cells, despite these markers being transcriptionally regulated in parallel with IgD as part of the B-cell maturation program. CD23 and CD21 are induced during B-cell maturation by BAFF-receptor signaling to NF-κB transcription factors, whereas IgD expression does not depend on this transcriptional regulatory system (27, 28). It will be interesting to see whether there are other Zfp318-dependent genes through global gene expression analyses in the mutant B cells.

The similarities and differences between the Zfp318 point mutant and null mutant have several implications. In mice with the point mutation, follicular B cells retained substantial IgD expression, whereas IgD was almost fully extinguished by the null mutation. Because the point mutation is within the alternatively spliced exon 10 and only alters the mRNA and protein sequence of the ZFP318 long isoform, the residual IgD-promoting activity may reflect action of the short isoform. However, the fact that the point mutation fully extinguished IgD expression on marginal zone B cells and decreased IgD on follicular B cells shows that the long isoform has the major role in promoting normal IgD expression. In cell transfection studies, the long and short ZFP318 isoforms have been shown to have opposite stimulatory and inhibitory effects, respectively, on transcription induced by the androgen receptor (20). Hence, it is possible that only the ZFP318 long form promotes IgD expression and the point mutation cripples, but does not abolish, its activity, so that residual IgD expression occurs only in follicular B cells with the highest Zfp318 mRNA levels. The point mutation changes an isoleucine in the long isoform that is conserved from fish to humans within an unannotated domain marked by 16 lysine residues that are also highly conserved. Based on the effects of the point mutation, we propose to call this conserved region the LRID domain. As occurs in well-characterized RNA-binding domains, the charged lysine residues in the LRID domain may bind RNA and cooperate with RNA binding by the two U1-type zinc finger domains in the ZFP318 long isoform.

Although it is possible that ZFP318 controls IgD and IgM expression at the level of protein trafficking or by inhibiting expression or activity of mRNA polyadenylation cleavage enzymes, several lines of evidence favor a simpler hypothesis that ZFP318 directly regulates alternative RNA splicing of Ighm and Ighd constant region exons. First, differential expression of IgD during B-cell maturation has been shown to occur at the level of mRNA production (12, 13, 29⇓–31) Second, the ZFP318 zinc fingers are of the U1 type defined by the RNA-binding zinc finger in the spliceosomal U1C protein (25, 32) and of the zf-C2H2_JAZ superfamily that bind double-stranded RNA or RNA-DNA hybrids (33). Third, both the long and short isoforms of ZFP318 accumulate selectively in the nucleus, with the short isoform localized to subnuclear speckles that contain histone deacetylase 2 (HDAC2) and are adjacent to nuclear speckles containing serine/arginine-rich splicing factor 2 (SRSF2, SC-35; refs. 18–21). ZFP318 binding to HDAC2 (21) is potentially similar to the recently demonstrated interaction between the RNA-binding protein HuR and HDAC2 to influence alternative mRNA splicing (34). A large amount of alternative splicing occurs cotranscriptionally when pre-mRNAs are still chromatin associated, where it is governed by two-way cooperation between RNA-binding splicing factors such as HuR or SRSF proteins, the extent of histone acetylation along intragenic chromatin, and the speed of RNA polymerase II (Pol II) transcript elongation along chromatin (34⇓⇓⇓–38).

The evidence above, combined with earlier models for Ighd expression (12, 13, 31), leads us to propose the following simple hypothesis for ZFP318-dependent IgD expression (Fig. S1). (i) The rate of VDJH exon splicing to Ighd competes with the rate of Igh pre-mRNA cleavage at the Ighm polyadenylation site. Because the latter is located 5′ to Ighd on the pre-mRNA, if cleavage occurs first, it precludes VDJH splicing to Ighd. (ii) In the absence of ZFP318, Pol II elongates the Ighm-Ighd pre-mRNA at a slower rate than polyadenylation site cleavage, so that most pre-mRNAs are cleaved at the Ighm polyadenylation site before Pol II has transcribed the Ighd exons. (iii) In mature B cells, ZFP318 is recruited to the Igh pre-mRNA via its U1-type zinc fingers and its LRID domain and associates with and inhibits HDAC2, thereby promoting hyperacetylation of Igh chromatin and more rapid Pol II elongation of the Ighm-Ighd pre-mRNA. When the rate of Igh pre-mRNA elongation exceeds the rate of polyadenylation site cleavage, a substantial fraction of Ighd exons are now spliced to VDJH. Variations to this hypothesis should nevertheless also be considered, including the possibility that ZFP318 binds particular sites in Igh pre-mRNA to suppress recognition of Ighm splice acceptors or polyadenylation sites or to enhance spliceosomal recognition of Ighd splice acceptors.

Testing these hypotheses in the future can be facilitated by the Zfp318 mouse mutants described here, for example by ChipSeq experiments to define ZFP318 binding sites and measurement of histone acetylation and pol II elongation rates in the Igh locus of mutant and wild-type B cells. Overall, the findings here reveal the function of a regulator of B-cell maturation and open up avenues to understand the regulation of alternative mRNA splicing.

Materials and Methods

Mouse Strains and Procedures.

The Zfp318 point mutant strain was identified by flow cytometry screening of peripheral blood lymphocytes in third-generation offspring from ENU-treated C57BL/6 mice as described (39). Identification of the causal mutation was done by whole exome sequencing as described (40, 41). The Zfp318−/− mice were generated by H.H. by inserting a GFP-Neomycin cassette into exon 2 of the Zfp318 gene. Knock-out mice on a C57BL/6 background were obtained from the RIKEN BioResource Center (RBRC01768). All animals were housed under specific pathogen-free conditions at the Australian Phenomics Facility. All animal experiments were approved by the Australian National University Animal Ethics and Experimentation Committee.

FACS.

Lymphocytes from blood, spleen, and bone marrow were prepared, stained, and analyzed by flow cytometry according to published methods (42). Samples were analyzed by using a BD LSR II or LSRFortessa flow cytometer.

Microarray Analysis.

IgD−IgM−CD43intCD24int Pro-B cells were sorted from the bone marrow, and CD93−IgMlow mature and CD93highIgMhigh immature B cells were sorted from the spleen of naive wild-type C57BL/6 mice by flow cytometry. Isolated cells were pelleted and snap frozen in liquid nitrogen before shipment to Miltenyi Biotech Genomic Services (Bergisch Gladbach) for RNA extraction and global gene expression analysis by Agilent single color 8 × 60K Whole Mouse Genome Microarray.

Acknowledgments

We thank the staff the Australian Phenomics Facility for excellent animal husbandry and the staff of the John Curtin School of Medical Research Microscopy and Cytometry Resource Facility. We also thank Nadine Barthel for excellent technical assistance. This work was supported by National Institutes of Health Grant U19 AI100627, a Ramaciotti Foundation grant (to A.E. and C.C.G.), and National Health and Medical Research Council of Australia Grants 585490, 1016953 (to C.G.G.), and 1035858 (to A.E.).

Footnotes

  • ↵1To whom correspondence may be addressed. E-mail: chris.goodnow{at}anu.edu.au or anselm.enders{at}anu.edu.au.

  • Author contributions: A.E., A.S., L.A.M., H.B., Y.S., E.M.B., B.W., B.B., G.S., M.A.F., T.D.A., and C.C.G. designed research; A.E., A.S., L.A.M., H.B., Y.S., B.W., B.B., G.S., M.A.F., T.D.A., and C.C.G. performed research; K.Y. and H.H. contributed new reagents/analytic tools; A.E., A.S., L.A.M., H.B., Y.S., E.M.B., B.W., B.B., G.S., M.A.F., T.D.A., and C.C.G. analyzed data; A.E. and C.C.G. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1402739111/-/DCSupplemental.

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ZFP318 is essential for IgD expression
Anselm Enders, Alanna Short, Lisa A. Miosge, Hannes Bergmann, Yovina Sontani, Edward M. Bertram, Belinda Whittle, Bhavani Balakishnan, Kaoru Yoshida, Geoff Sjollema, Matthew A. Field, T. Daniel Andrews, Hiromi Hagiwara, Christopher C. Goodnow
Proceedings of the National Academy of Sciences Mar 2014, 111 (12) 4513-4518; DOI: 10.1073/pnas.1402739111
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