Zinc-Finger Protein ZFP318 Is Essential for Expression of Igd, the Alternatively Spliced Igh Product Made by Mature B Lymphocytes

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

Anselm Endersa,1, Alanna Shorta, Lisa A. Miosgea, Hannes Bergmanna, Yovina Sontania, Edward M. Bertrama,b, Belinda Whittleb, Bhavani Balakishnanb, Kaoru Yoshidac, Geoff Sjollemab, Matthew A. Fielda, T. Daniel Andrewsa,b, Hiromi Hagiwarac, and Christopher C. Goodnowa,1

aDepartment of Immunology and bAustralian Phenomics Facility, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia; and cDepartment of Biomedical Engineering, Toin University of Yokohama, Aoba-ku, Yokohama 225-8503, Japan

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

IgD and IgM are produced by alternative splicing of long primary of separate LHV, D, and J elements in pre-B cells. Downstream RNA transcripts from the Ig heavy chain (Igh) locus and serve as from the VDJH exon are six Ighm constant region exons encoding the receptors for antigen on naïve mature B lymphocytes. IgM is the extracellular and transmembrane segments of membrane IgM, made selectively in immature B cells, whereas IgD is coexpressed then five Ighd constant region exons encoding the corresponding with IgM when the cells mature into follicular or marginal zone B segments of IgD, and finally similar sets of Ighg, Ighe,orIgha exons cells, but the transacting factors responsible for this regulated encoding the constant regions of IgG, IgE, and IgA. Isotype change in splicing have remained elusive. Here, we use a genetic switching results from further DNA recombination within the locus screen in mice to identify ZFP318, a nuclear protein with two U1-type that deletes the Ighm and Ighd exons and brings either the Ighg, Ighe Igha ′ VDJ zinc fingers found in RNA-binding proteins and no known role in ,or exons immediately 3 to the H exon, so that the latter is spliced to IgG, IgE, or IgA constant region exons in the the immune system, as a critical factor for IgD expression. A point resulting mRNA (6–8). IgD is the exception, however, because most mutation in an evolutionarily conserved lysine-rich domain B cells do not express IgD by DNA recombination but instead encoded by the alternatively spliced Zfp318 exon 10 abolished via a reversible, developmentally regulated process of alterna- IMMUNOLOGY IgD expression on marginal zone B cells, decreased IgD on follic- tive mRNA splicing of the VDJH exon to the Ighm and Ighd exons ular B cells, and increased IgM, but only slightly decreased the (5, 9, 10). This unique arrangement for coexpression of IgM and percentage of B cells and did not decrease expression of other IgD mRNA by alternative splicing is conserved in bony fish, maturation markers CD21, CD23, or CD62L. A targeted Zfp318 amphibians, reptiles, monotremes, and mammals (11), yet it is not null allele extinguished IgD expression on mature B cells and known how IgD mRNA is selectively produced in mature B cells. increased IgM. Zfp318 mRNA is developmentally regulated in Pre-B cells and immature B cells express very little IgD parallel with IgD, with little in pro-B cells, moderate amounts mRNA and express only IgM, despite transcribing the Ighd exons in immature B cells, and high levels selectively in mature follicu- at levels that are often only two- to threefold lower than the Ighm + − + lar B cells. These findings identify ZFP318 as a crucial factor reg- exons and not differing between IgD and IgD IgM B cells, ulating the expression of the two major antibody isotypes on the when measured by RNA-polymerase run-on experiments in surface of most mature B cells. isolated nuclei (12–16). These results have led to the hypothesis that 25-kb-long Igh pre-mRNA transcripts traverse from the IgHm | IgHd | immunoglobulin isotype | ENU mutation VDJH exon through the Ighd exons in both immature and mature

g isotypes with different heavy (H)-chain constant regions are Significance Imade by B lymphocytes in a developmentally regulated series (1). The different antibody isotypes serve as cell surface markers Mammalian B lymphocytes make antibodies of five different of B-cell maturation, as functionally distinct receptors for B-cell heavy chain isotypes, IgM, IgD, IgG, IgE, and IgA. The different activation by antigens and as secreted mediators of different anti- isotypes are produced at discrete stages in B-cell development body effector functions (2). All B cells begin as immature B cells in from a single immunoglobulin heavy chain (Igh) gene, either by bone marrow or fetal liver that express only the IgM isotype on their irreversible rearrangement of the gene to make IgG, IgE, or IgA cell surface (3), comprised of H chains with an N-terminal variable or by alternative splicing of the RNA transcribed from the Igh domain and C-terminal constant region domains, transmem- gene to coexpress IgM and IgD. Developmentally regulated brane segment, and cytoplasmic tail, paired with Ig light chains. trans-acting factors have been hypothesized to control IgM Maturation into follicular B cells, which recirculate among the and IgD expression from large Igh RNAs, but these factors have spleen, lymph nodes, and other secondary lymphoid tissues, is remained elusive for several decades. Here, using a genome- marked by coexpression of a second isotype, IgD. Each mature wide mutation screen in mice, we identify an obscure gene, follicular B cells displays a mixture of cell surface B-cell receptors Zfp318, as encoding a specific and essential factor promoting (BCRs) comprising the same variable domain joined to either IgD IgD expression in mature B cells. or IgM constant regions, with greater levels of IgD than IgM (4, 5). B cells undergo isotype switching after activation by microbial 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., antigens and helper T cells: They irreversibly lose IgM and IgD and and C.C.G. performed research; K.Y. and H.H. contributed new reagents/analytic tools; switch to expressing the same variable domain linked to IgG, IgA, 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 or IgE constant region domains. Although the process of isotype data; A.E. and C.C.G. wrote the paper. switching to IgG, IgA, and IgE is well understood, the mechanism The authors declare no conflict of interest. for developmentally regulated IgD expression remains obscure. Freely available online through the PNAS open access option. The developmental order of antibody isotype expression is 1To whom correspondence may be addressed. E-mail: [email protected] or reflected in the layout of the Ig heavy chain locus, Igh.Insurface [email protected]. + IgM immature B cells, transcription begins with two variable This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. exons (LH and VDJH) formed by intrachromosomal recombination 1073/pnas.1402739111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1402739111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 B cells, but an unknown transacting factor alters splicing either by decreased IgD and increased IgM on mature B cells (Fig. 1 A by: (i) promoting RNA cleavage at Ighm polyadenylation sites in and B). Homozygotes had one-third as much IgD as wild-type immature B cells to preclude VDJH splicing to Ighd;or(ii) si- littermates and unrelated controls, whereas heterozygotes had an lencing Ighm cleavage and polyadenylation sites in mature B ∼25% decrease. The frequency of B cells was slightly reduced cells to allow splicing to Ighd (13). In some immature B cells, (Table 1). Exome sequencing of an affected animal followed by failure to express IgD also appears to reflect unloading of genotyping of candidate mutations in a large cohort of siblings RNA Pol II at an attenuation region 3′ to Ighm and 5′ to Ighd, and offspring revealed that the low IgD trait was completely cor- but when this region is removed, there is still little splicing to related with inheritance of a point mutation in the gene encoding IgD (16, 17). In contrast, in terminally differentiated plasma zinc-finger protein (ZFP) 318. cells, transcription termination occurs upstream of Ighd,re- ZFP318 is also called testicular zinc-finger protein (TZF) and sulting in very low expression of Ighd mRNA. has been implicated in transcriptional regulation in testes with Although differential expression of IgM and IgD was one of the genetic deficiency causing infertility in mice (18–21) but has no first examples of developmentally regulated alternative mRNA known function in the immune system. Microarray comparisons splicing, progress to understand its basis has stalled because it has of gene expression in B-cell subsets have identified Zfp318 as not been possible to identify the nature of the transacting factors. a member of a set of mRNAs that increases during maturation of Here, we use a phenotype-driven genetic screen in mice to identify immature B cells into mature follicular B cells (22–24). By ana- a gene that fulfils the criteria for encoding the elusive trans- lyzing flow-sorted B-cell subsets, we confirmed expression of activating factor promoting IgD expression. Zfp318 mRNA closely parallels IgD heavy chain (Ighd) mRNA during B-cell development (Fig. 1C). There was very little Zfp318 Results in pro-B cells in the bone marrow, moderate amounts in imma- + − Identification of a Missense Mutation in Zfp318 Causing Decreased ture (CD93 , CD62L ) B cells in the spleen, and high amounts in − + IgD and Increased IgM. In a peripheral blood screen of mice inher- mature follicular (CD93 , CD62L ) B cells. The close correlation iting ethylnitrosourea (ENU)-induced point mutations, we identi- between IgD and Zfp318 expression is reinforced in the IMM- fied a pedigree with a Mendelian recessive mutation characterized GEN dataset (22, 23), which shows highest Zfp318 expression in

Lymphocytes ABwild-type homozygote D Ile1347Thr 5 30 8 CCDS 28827.2 Zfp318 long 2237 aa

10 3 3 6 104 20.4 30.9 20 123456789 10 103 64 57.2 4 10 CCDS 28826.2 Zfp318 short 1154 aa 102 2 IgD MFI x 10 0 IgM MFI x 10 CD3 0 0 2 3 4 5 123456711 010 10 10 10 wt het hom wt het hom CD19 C U1-type C2H2 zinc finger B cells wild-type homozygote ) 2 5 6 5 Lysine-rich IgD-promoting domain 10 ) 87.2 76.3 3 4 104 3 4 Proline-rich domain

3 (a.u. x 10 10 2 2 102 1 0 IgD Zfp318 0 Ighd (a.u. x 10 0 0102 103 104 105

IgM pro-B pro-B mature mature E immature immature

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.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402739111 Enders et al. Downloaded by guest on September 30, 2021 − − Table 1. Frequency of B-cell subpopulations in bone marrow and spleen in Zfp318 point mutant mice and in the blood of Zfp318 / Immature Mature Transitional Follicular Genotype Organ B cells Pro-B cells Pre-B cells B cells B cells B cells B cells MZ B cells No. of animals

WT BM 21.1 ± 2.7 9.0 ± 1.5 22.1 ± 0.6 15.1 ± 1.0 36.5 ± 0.4 ——— 5 Het BM 20.4 ± 2.1 8.4 ± 0.9 18.7 ± 3.2 14.2 ± 0.9 39.6 ± 3.2 ——— 6 Hom BM 21.1 ± 2.0 7.4 ± 2.4 17.2 ± 3.2 12.6 ± 2.5 45.2 ± 7.6 ——— 4 WT Spleen 57.9 ± 3.9 ——— —25.5 ± 7.0 56.0 ± 4.7 10.9 ± 3.4 9 Het Spleen 56.1 ± 4.6 ——— —25.4 ± 6.8 56.9 ± 4.7 10.3 ± 2.1 10 Hom Spleen 51.0 ± 3.0 ——— —21.9 ± 7.2 59.6 ± 5.2 11.4 ± 2.8 7 Zfp318+/+ Blood 62.0 ± 4.8 ——21.4 ± 5.1 71.7 ± 6.7 ——— 4 + − Zfp318 / Blood 57.9 ± 5.1 ——17.5 ± 4.2 74.6 ± 5.0 ——— 22 − − Zfp318 / Blood 53.5 ± 4.2 ——19.1 ± 6.1 69.8 ± 7.4 ——— 15

The frequencies are expressed as mean values ± SD out of all live cells (B cells) or as a percentage of all B cells for all other populations. The last column shows the number of animals analyzed per genotype analyzed in one (BM and blood) or two (spleen) experiments. Statistical analysis was done comparing all groups within one organ by one-way ANOVA followed by Bonferroni post hoc test to compare each population in the heterozygous and homozygous mice to + the relevant WT control. Statistically significant differences are marked in bold. B cells, B220 in live lymphocytes gate for BM and in lymphocyte gate for + + − − + − − + + − − spleen. Immature B cells, IgM CD93 ; mature B cell, CD93 ; pre-B cell, CD24hiCD43 CD93 IgD IgM ; pro-B cell, CD24intCD43 CD93 IgD IgM ; in the bone − − − + + marrow. Follicular, CD93 CD23hiCD21med; marginal zone B cells, CD93 CD23 CD21hi; transitional B cells, CD93 ; in the spleen and total B cells (CD19 ), + − − − + − + + immature (CD93 ), and mature (CD93 ) B cells in the blood of Zfp318 / mice as well as Zfp318 / and Zfp318 / littermate controls.

IgDhigh follicular B cells and T3 transitional B cells, lower Zfp318 consequently, encodes a protein of 1,154 aa lacking the second in IgDlow marginal zone B cells and T1-T2 transitional B cells, zinc finger and the polyproline domain. The IgD-lowering mu- + and very low expression in IgD-negative IgM immature B cells in tation was a nonsynonymous T > C transition in the differentially the bone marrow. spliced exon 10. Because this mutation only alters the mRNA and Two alternatively spliced isoforms of Zfp318 mRNA are an- protein sequence of the long form, it demonstrates that the long

notated in the genome and described in the literature (18) (Fig. Zfp318 isoform promotes normal IgD expression. The mutation IMMUNOLOGY 1D). The long isoform (CCDS 28827.2) includes exons 1–10 and changed codon 1347 in the long isoform from a highly conserved encodes a 2,237-aa protein containing two C2H2 zinc-finger hydrophobic isoleucine into a polar threonine. Ile1347 lies in an domains of the U1 ribonucleoprotein type (25). A 44-aa linker unannotated domain between the second zinc finger and the separates the two zinc fingers, similar to the 34- to 44-aa linkers polyproline domain, containing 16 lysine residues that are highly between the dsRNA-binding U1-type zinc fingers in the JAZ and conserved in mammals, birds, reptiles, and bony fish (Fig. 1E). ZFa proteins and unlike the 6- to 8-aa linkers typically present We refer to this domain as the lysine-rich IgD-promoting domain between C2H2 zinc fingers in DNA binding proteins (26). The (LRID). It is notable that the conserved Ile residue is substituted short isoform (CCDS 28826.2) skips exons 8, 9, and 10 and, to Glu in Maylandia zebra and other Cichlid fish species,

A Live cells B220+ cells CD93-CD19+ B cells CD93+ B cells precursor B cells 25.1 35.9 52.4 64.7 26.5

WT 64.4

3.6

5 23.7 5 5 5 5 10 10 10 10 10 64.7 27.2 38.9 47.6 104 104 104 104 104 Hom 103 103 103 103 64.7 103

102 102 102 102 102 0 0 0 0 0 4.3 0 100k 200k 0102 103 104 105 0100 102 103 104 105 0100 102 103 104 105 0100 102 103 104 105 B220 CD19 IgD IgD CD24

FSC CD93 IgM IgM CD43

B Immature B cells Mature B cells ns # # # 60 ns ns 4 ns *** 80 * # 25 ** # Fig. 2. B-cell development in bone marrow of 20 3 60 Zfp318 point mutant mice. (A) Flow cytometry of 2 2 2 3 40 bone marrow lymphocytes from Zfp318 mutant mice 15 (Hom) and wild-type littermate controls (WT). B cells 2 40 were gated as B220+ cells. Expression of IgM and IgD + − 10 was analyzed on CD93 immature and CD93 mature IgD MFI x 10 IgM MFI x 10 IgM MFI x 10 20 IgD MFI x 10 Bcells.(B) MFI of IgM and IgD on immature and 1 20 5 mature B cells in the bone marrow gated as in A. Statistical analysis by one-way ANOVA followed by 0 0 0 0 Bonferroni post hoc test: ns, P > 0.05; *P < 0.05; **P < WT Het Hom WT Het Hom WT Het Hom WT Het Hom 0.01; ***P < 0.001; #P < 0.0001.

Enders et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 # A Lymphocytes B220+ B cells CD93- B cells CD93+ B cells B 25 ns # 14.1

35.2 64.5 31.1 3 78.5 20 55.8 # # 15 WT ns # *# 10 IgM MFI x 10

5 105 105 105 105 42.3 62.6 32.4 14.5 79.3 0 104 104 104 104 Hom 47.1 WT Het Hom WT Het Hom WT Het Hom Immature Follicular Marginal zone 103 103 103 103 20 # 2 2 2 2 10 10 10 10 ## 0 0 0 0 3 15 0100 102 103 104 105 0100 102 103 104 101 5 0010102 103 104 101 5 0100 102 103 104 105 # # TCRβ CD62L CD21 IgD 10 ** # ##

B220 CD93 CD23 IgM IgD MFI x 10 5 C ns ns ns 15 ns ns ** 4 0 2 ns ns ns ns

ns ** 3

3 3 3 WT Het WT Het WT Het 3 4 Hom Hom Hom 10 Immature Follicular Marginal zone 2 2 Follicular B cells 5 2 1 1 BAFF-R MFI x 10 CD62L MFI x 10 CD62L CD21 MFI x 10 0 CD23 MFI x 10 0 0 0 WT Het Hom WT Het Hom WT Het Hom WT Het Hom ns ns 15 15 ns ns 2 ns ns

ns ns 3

2 3 ns ns ns ns 3 1 10 10 MZ B cells 2 5 0.5 1 5 CD62L MFI x 10 CD62L CD23 MFI x 10 BAFF-R MFI x 10 0 0 CD21 MFI x 10 0 0 WT Het Hom WT Het Hom WT Het Hom WT Het Hom

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)MFIof 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.

although we are unaware of any data on IgD expression in events were comparable in Zfp318I1347T homozygous mutant these species. and wild-type mice. The two main subsets of mature B cells in the + − spleen, CD23 CD21med follicular B cells and CD23 CD21hi I1347 Effect of Zfp318 Mutation on B-Cell Developmental Subsets in marginal-zone B cells, were both present in normal frequencies Bone Marrow and Spleen. Analysis of B-cell development in the (Fig. 3A and Table 1). The lower level of IgD expressed on bone marrow of Zfp318 mutant mice and littermate controls wild-type marginal zone B cells was completely extinguished showed no significant difference in either percentage of B cells in Zfp318I1347T homozygotes, representing a decrease of ∼10-fold or subset distribution of developing B cells but a small increase on marginal zone B cells compared with only ∼threefold decreased in the proportion of mature recirculating B cells (Fig. 2A and IgD on follicular B cells (Fig. 3B). + + Table 1). Interestingly, the subset of CD93 , IgM immature B cells in the bone marrow that starts to express IgD at very low Loss of IgD Expression in Mice with a Zfp318 Null Mutation. The levels already showed a reduced expression of IgD compared above finding that IgD expression was extinguished on marginal with either wild-type (P < 0.0001) or heterozygous (P < 0.001) zone B cells but persisted at moderate levels on homozygous littermate controls without a statistically significant change of mutant follicular B cells had two alternative explanations: (i) IgM expression (Fig. 2B). In contrast, the mature, recirculating B another gene might also contribute to IgD expression in follic- cells in the bone marrow had reduced expression of IgD and ular B cells; or (ii) the Zfp318I1347T point mutation may only increased expression of IgM (Fig. 2B). partially compromise ZFP318 function. To resolve these alter- Splenic B cells in homozygous mutants showed a similarly de- natives, we analyzed circulating B cells in mice carrying a null creased IgD and increased IgM, but normal expression of other Zfp318 allele generated by a targeted insertion in exon 2. Homo- mature B-cell surface markers with the exception of slightly in- zygous Zfp318 null mice had a normal percentage of circulating creased CD23 (Fig. 3 A–C). In normal B cells, CD93 expression is B cells and a normal fraction of these B cells that had matured to extinguished, whereas CD62L, CD23, CD21, and BAFF-Receptor theCD93-negativestage(Fig.4A), but IgD expression was almost are induced when immature B cells in the spleen mature into re- completely abolished on immature and mature B cells (Fig. 4 circulating follicular B cells, and these developmentally regulated B and C). IgM was increased threefold on mature B cells from

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402739111 Enders et al. Downloaded by guest on September 30, 2021 B The similarities and differences between the Zfp318 point A Lymphocytes B cells CD19+ B cells mutant and null mutant have several implications. In mice with 76 21.9 the point mutation, follicular B cells retained substantial IgD 25.7 expression, whereas IgD was almost fully extinguished by the null +/+ 66.3 mutation. Because the point mutation is within the alternatively spliced exon 10 and only alters the mRNA and protein sequence 010 1010 10 of the ZFP318 long isoform, the residual IgD-promoting activity IgM may reflect action of the short isoform. However, the fact that the point mutation fully extinguished IgD expression on marginal 10 10 30.4 78.2 20 zone B cells and decreased IgD on follicular B cells shows that 10 10 the long isoform has the major role in promoting normal IgD ex- -/- 61.7 pression. In cell transfection studies, the long and short ZFP318 10 10 010 1010 10 isoforms have been shown to have opposite stimulatory and in- 10 10 IgD hibitory effects, respectively, on transcription induced by the an- 0 0 -/- CD3 Zfp318 T cells drogen receptor (20). Hence, it is possible that only the ZFP318 -/- 010 1010 10 CD19 010 1010 10 Zfp318 B cells +/+ long form promotes IgD expression and the point mutation crip- CD19 CD93 Zfp318 B cells ples, but does not abolish, its activity, so that residual IgD expression occurs only in follicular B cells with the highest Zfp318 C immature B cells mature B cells 2.5 15 15 mRNA levels. The point mutation changes an isoleucine in the 10 long isoform that is conserved from fish to humans within an 2.0 10 10 unannotated domain marked by 16 lysine residues that are also 1.5 highly conserved. Based on the effects of the point mutation, we 5 1.0 propose to call this conserved region the LRID domain. As 5 5 IgM MFI x 10 IgD MFI x 10 IgM MFI x 10 IgD MFI x 10 0.5 occurs in well-characterized RNA-binding domains, the charged lysine residues in the LRID domain may bind RNA and co- 0 0 0 0 +/++/- -/- +/++/- -/- +/+ -/-+/- +/+ -/-+/- operate with RNA binding by the two U1-type zinc finger domains in the ZFP318 long isoform. Fig. 4. Loss of IgD expression on circulating B cells of mice with a Zfp318 Although it is possible that ZFP318 controls IgD and IgM null mutation. (A) Representative flow cytometry of peripheral blood lym- expression at the level of protein trafficking or by inhibiting ex- IMMUNOLOGY phocytes from a Zfp318 homozygous null (−/−) and wild-type littermate + + + + pression or activity of mRNA polyadenylation cleavage enzymes, control ( / ) showing the frequency of CD3 T cells and CD19 B cells among − + several lines of evidence favor a simpler hypothesis that ZFP318 lymphocytes (Left), and the frequency of CD93 mature and CD93 imma- + directly regulates alternative RNA splicing of Ighm and Ighd ture B cells among CD19 lymphocytes (Right). (B) Expression of IgM (Upper) − − and IgD (Lower) on B cells from Zfp318 homozygous null ( / , black line) and constant region exons. First, differential expression of IgD dur- wild-type (+/+, shaded). As a negative control, staining on T cells from ing B-cell maturation has been shown to occur at the level of − − + – Zfp318 / mice is also shown (dashed line). (C) MFI of IgM and IgD on CD93 mRNA production (12, 13, 29 31) Second, the ZFP318 zinc − − − immature and CD93 mature B cells from Zfp318 / mice compared with fingers are of the U1 type defined by the RNA-binding zinc heterozygous and wild-type littermate controls. 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 homozygous Zfp318 null mice. Thus, ZFP318 is absolutely of ZFP318 accumulate selectively in the nucleus, with the short required for IgD expression and the point mutation only isoform localized to subnuclear speckles that contain histone partially inactivates its function. deacetylase 2 (HDAC2) and are adjacent to nuclear speckles containing serine/arginine-rich splicing factor 2 (SRSF2, SC-35; Discussion refs. 18–21). ZFP318 binding to HDAC2 (21) is potentially The findings above identify ZFP318 as a long-sought transacting similar to the recently demonstrated interaction between the factor governing differential expression of IgM and IgD isotypes RNA-binding protein HuR and HDAC2 to influence alternative during B-cell maturation. Earlier work demonstrated that 25-kb- mRNA splicing (34). A large amount of alternative splicing long heavy chain pre-mRNAs are transcribed from the VDJH occurs cotranscriptionally when pre-mRNAs are still chromatin exon through the Ighm and Ighd exons in both immature and associated, where it is governed by two-way cooperation between mature B cells (12–16). Consequently, it was hypothesized that RNA-binding splicing factors such as HuR or SRSF proteins, the the alternative splice acceptor sites on the first Ighm and Ighd extent of histone acetylation along intragenic chromatin, and the constant region exons must compete for the single splice donor speed of RNA polymerase II (Pol II) transcript elongation along – sequence in the VDJH exon, with unknown transacting factors chromatin (34 38). either suppressing RNA splicing to the Ighd exons in immature B The evidence above, combined with earlier models for Ighd cells or promoting Ighd splicing in mature B cells at the expense expression (12, 13, 31), leads us to propose the following simple of Ighm. The results here support the latter hypothesis: Zfp318 hypothesis for ZFP318-dependent IgD expression (Fig. S1). (i) mRNA increases during B-cell maturation in parallel with in- The rate of VDJH exon splicing to Ighd competes with the rate of creasing IgD, and Zfp318 mutations increase IgM and extinguish Igh pre-mRNA cleavage at the Ighm polyadenylation site. Be- IgD expression. cause the latter is located 5′ to Ighd on the pre-mRNA, if Zfp318 appears to be specifically required for balancing IgD cleavage occurs first, it precludes VDJH splicing to Ighd.(ii)In and IgM output from Igh, and not for the overall program of the absence of ZFP318, Pol II elongates the Ighm-Ighd pre- B-cell maturation. Zfp318 mutation had no effect on the accu- mRNA at a slower rate than polyadenylation site cleavage, so mulation of mature B-cell subsets nor did it diminish the ex- that most pre-mRNAs are cleaved at the Ighm polyadenylation pression of CD21, CD62L, or CD23 on mature B cells, despite site before Pol II has transcribed the Ighd exons. (iii) In mature B these markers being transcriptionally regulated in parallel with IgD cells, ZFP318 is recruited to the Igh pre-mRNA via its U1-type as part of the B-cell maturation program. CD23 and CD21 are zinc fingers and its LRID domain and associates with and inhibits induced during B-cell maturation by BAFF-receptor signaling to HDAC2, thereby promoting hyperacetylation of Igh chromatin NF-κB transcription factors, whereas IgD expression does not de- and more rapid Pol II elongation of the Ighm-Ighd pre-mRNA. pend on this transcriptional regulatory system (27, 28). It will be When the rate of Igh pre-mRNA elongation exceeds the rate interesting to see whether there are other Zfp318-dependent genes of polyadenylation site cleavage, a substantial fraction of Ighd through global gene expression analyses in the mutant B cells. exons are now spliced to VDJH. Variations to this hypothesis

Enders et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 should nevertheless also be considered, including the possibility were approved by the Australian National University Animal Ethics and that ZFP318 binds particular sites in Igh pre-mRNA to suppress Experimentation Committee. recognition of Ighm splice acceptors or polyadenylation sites or to enhance spliceosomal recognition of Ighd splice acceptors. FACS. Lymphocytes from blood, spleen, and bone marrow were prepared, Testing these hypotheses in the future can be facilitated by the stained, and analyzed by flow cytometry according to published methods Zfp318 mouse mutants described here, for example by ChipSeq (42). Samples were analyzed by using a BD LSR II or LSRFortessa flow experiments to define ZFP318 binding sites and measurement of cytometer. histone acetylation and pol II elongation rates in the Igh locus − − of mutant and wild-type B cells. Overall, the findings here Microarray Analysis. IgD IgM CD43intCD24int Pro-B cells were sorted from − reveal the function of a regulator of B-cell maturation and the bone marrow, and CD93 IgMlow mature and CD93highIgMhigh immature open up avenues to understand the regulation of alternative B cells were sorted from the spleen of naive wild-type C57BL/6 mice by flow mRNA splicing. cytometry. Isolated cells were pelleted and snap frozen in liquid nitrogen before shipment to Miltenyi Biotech Genomic Services (Bergisch Gladbach) Materials and Methods for RNA extraction and global gene expression analysis by Agilent single × Mouse Strains and Procedures. The Zfp318 point mutant strain was identified color 8 60K Whole Mouse Genome Microarray. by flow cytometry screening of peripheral blood lymphocytes in third-gen- eration offspring from ENU-treated C57BL/6 mice as described (39). Identi- ACKNOWLEDGMENTS. We thank the staff the Australian Phenomics Facility fication of the causal mutation was done by whole exome sequencing as for excellent animal husbandry and the staff of the John Curtin School of − − Medical Research Microscopy and Cytometry Resource Facility. We also described (40, 41). The Zfp318 / mice were generated by H.H. by inserting thank Nadine Barthel for excellent technical assistance. This work was a GFP-Neomycin cassette into exon 2 of the Zfp318 gene. Knock-out mice on supported by National Institutes of Health Grant U19 AI100627, a Ramaciotti a C57BL/6 background were obtained from the RIKEN BioResource Center Foundation grant (to A.E. and C.C.G.), and National Health and Medical (RBRC01768). All animals were housed under specific pathogen-free Research Council of Australia Grants 585490, 1016953 (to C.G.G.), and conditions at the Australian Phenomics Facility. All animal experiments 1035858 (to A.E.).

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