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Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1

Nature Immunology volume 18, pages 683–693 (2017)Cite this article

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Abstract

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3′ untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

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Figure 1: MZ B cells specifically require ZFP36L1.
Figure 2: B cell–intrinsic requirement for ZFP36L1.
Figure 3: ZFP36L1 is selectively required for the maintenance of MZ and MZP B cells.
Figure 4: Gene-expression analysis following deletion of Zfp36l1.
Figure 5: ZFP36L1 enforces MZ B cell identity.
Figure 6: ZFP36L1 regulates IRF8 expression.
Figure 7: ZFP36L1 regulates KLF2.
Figure 8: ZFP36L1 controls the localization of MZ B cells in the splenic MZ.

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Acknowledgements

We thank M. Busslinger (Research Institute of Molecular Pathology) for the mice expressing CD23-Cre; M. Reth (Max Planck Institute for Immunobiology and Epigenetics) for the mice expressing Mb1-Cre; T. Ludwig (Columbia University) for the mice expressing R26ERT2−Cre; and D. Kioussis (National Institute for Medical Research) for the mice expressing CD2-iCre; K. Bates, R. Walker, A. Davies and L. Waugh and members of the Biological Services Unit for technical support; D. Bell and P. Tolar for help with immunofluorescence analysis; and members of the Turner laboratory, L. Webb, M. Linterman and T. Arnon for advice. Supported by a GlaxoSmithKline-CASE studentship, the Biotechnology and Biological Sciences Research Council, The Medical Research Council and Bloodwise.

Author information

Authors and Affiliations

  1. Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK

    Rebecca Newman, Helena Ahlfors, Alexander Saveliev, Alison Galloway, Robert Williams, Sarah E Bell & Martin Turner

  2. Immune Receptor Activation Laboratory, The Francis Crick Institute, London, UK

    Rebecca Newman

  3. Department of Haematology, University of Cambridge, The Clifford Allbutt Building, Cambridge Biomedical Campus, Cambridge, UK

    Daniel J Hodson

  4. School of Biosciences, University of Birmingham, Birmingham, UK

    Gurdyal S Besra

  5. MRC Centre for Immune Regulation, School of Immunity and Infection, University of Birmingham, Birmingham, UK

    Charlotte N Cook & Adam F Cunningham

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Contributions

R.N. designed and did most experiments; H.A. performed bioinformatics analysis; A.S. performed iCLIP analysis; A.G., D.J.H., R.W. and S.E.B. helped with mouse experiments; G.S.B. provided NP–α-GalCer; C.N.C. and A.F.C. performed some histology; and R.N. and M.T. planned the project and wrote the manuscript.

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Correspondence to Martin Turner.

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Integrated supplementary information

Supplementary Figure 1 Targeting strategy for the generation of Zfp36fl/fl mice.

(a) The Zfp36 gene consists of two exons (grey boxes). The targeting vector was designed to introduce loxP sites flanking exon 2, which contains the tandem zinc finger RNA binding domain, and a Neomycin selection cassette (neo) flanked by two FRT sites. The targeting construct was introduced into the Zfp36 locus of BRUCE4 embryonic stem cells (C57BL/6 origin) by homologous recombination. Positive clones were identified via Southern blotting confirming correct targeting at both 5’ and 3’ ends. Subsequent crossing of mice possessing the targeted allele to FlpE mice, removing the neo cassette generated the desired Zfp36fl/fl mice. The same gene targeting strategy was used to produce Zfp36l1fl/fl and Zfp36l2fl/fl mice (b) Introduction of the CD2-iCre transgene into homozygous Zfp36fl/fl mice caused a tissue specific deletion of exon 2, which was confirmed by Southern blotting.

Supplementary Figure 2 Peritoneal cavity B-1 cells require ZFP36L1, and the GFP–ZFP36L1 fusion protein is expressed from the transitional 2 B cell stage in R26GFPZFP36L1Cd23-Cre+ mice.

(a) Flow cytometry analysis of peritoneal cavity B cells from Zfp36l1fl/flMb1Cre/+ and Zfp36l1fl/flMb1+/+ mice. Numbers indicate percentage of cells within gate. Data represent at least 8 mice per genotype. (b) Number of B220loCD19+ B1 B cells and B220+CD19lo B2 B cells, in the peritoneal cavity as assessed by flow cytometry. (c) Flow cytometry analysis of peritoneal cavity B cells from Zfp36l1fl/flMb1Cre/+ and Zfp36l1fl/flMb1+/+ mice. Numbers indicate percentage of cells within gate. Dot plots are pre-gated B220intCD19hi cells. Data represent at least 8 mice per genotype. (d) Number of B220loCD19+CD5+IgM+ B1a B cells and B220loCD19+CD5-IgM+ B1b B cells, in the peritoneal cavity as assessed by flow cytometry. (e) Flow cytometry analysis of GFP-ZFP36L1 fusion protein expression in splenic B cell subsets from R26GFPZFP36L1Cd23-Cre+mice. Sample sizes: b, d (Zfp36l1fl/flMb1+/+ 10, Zfp36l1fl/flMb1Cre/+ 8). *P ≤ 0.05, **P ≤ 0.01 Mann-Whitney. Data pooled from 2 experiments (b, d; mean).

Supplementary Figure 3 MZ B cells require ZFP36L1 to persist over time.

(a) qPCR analysis showing relative Zfp36l1 exon 2 abundance in purified B cells from day 7 post tamoxifen treatment of Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/+ mice. (b) Absolute numbers of MZ B cells Fo B cells assessed by flow cytometry in mice as described in a. (c) qPCR analysis showing relative Zfp36l1 exon 2 abundance in purified B cells from day 10 post tamoxifen treatment of Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/+ mice. (d) Absolute numbers of MZ B cells Fo B cells assessed by flow cytometry in mice as described in c. (e) qPCR analysis showing relative Zfp36l1 exon 2 abundance in purified B cells from day 14 post tamoxifen treatment of Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/+ mice. (f) Absolute numbers of MZ B cells Fo B cells assessed by flow cytometry in mice as described in e. Sample sizes: a-f (4). *P ≤ 0.05, Mann-Whitney. Data from 1 experiment (a, c, e; mean + s.e.m.), data from 1 experiment (b, d, f; mean).

Supplementary Figure 4 ZFP36L1 controls turnover of MZ B cells.

(a) BrdU incorporation into splenic B cell subsets in Zfp36lfl/flMb1Cre/+ and Zfp36l1fl/flMb1+/+ mice fed BrdU in water for 14 days (b) BrdU incorporation into splenic B cell subsets from R26GFPZFP36L1Cd23-Cre+ and R26GFPZFP36L1Cd23-Cre- mice fed BrdU in water for 14 days. Sample sizes: a (Zfp36l1fl/flMb1+/+ 10, Zfp36lfl/flMb1Cre/+ 9), b (R26GFPZFP36L1Cd23-Cre- 4, R26GFPZFP36L1Cd23-Cre+ 6). *P ≤ 0.05, **P ≤ 0.01, Mann-Whitney. Data pooled from 2 independent experiments (a; mean), data from 1 experiment (b; mean).

Supplementary Figure 5 Validation of RNA-seq analysis and top regulated transcripts.

(a) Reads mapping to Zfp36l1. Reads were mapped using TopHat, and are displayed using IGV. Enrichment of uniquely aligning deduplicated reads shown in grey. The top 4 rows show reads in control (Zfp36l1+/+R26ERT2-Cre/ERT2-Cre) replicates, and the bottom 4 rows show reads in KO (Zfp36l1fl/flR26ERT2-Cre/+) replicates. (b) Normalised read counts for Zfp36 and Zfp36l2 in MZ B cells from tamoxifen treated Zfp36l1+/+R26ERT2-Cre/ERT2-Cre and Zfp36l1fl/flR26ERT2-Cre/+ mice. (c) A clustered heatmap showing the log2 transformed RPKM values of 50 most regulated transcripts based on their log2FoldChange in the comparison of Zfp36l1fl/flR26ERT2-Cre/+ MZ B cells to their Zfp36l1+/+R26ERT2-Cre/ERT2-Cre controls. Blue color indicates low, yellow medium and red high expressed genes. Dendrogram shows clustering of the genotypes. Sample sizes: a, b (4). Data from 1 experiment (b; mean + s.e.m.)

Supplementary Figure 6 Normal expression of p27 and Ki67 and DAPI profiles in ZFP36L1-deficient MZ B cells.

(a) Flow cytometry analysis showing p27 staining in MZ B cells from Zfp36l1fl/flMb1Cre/+ mice and Zfp36l1fl/flMb1+/+ controls (left panel) and tamoxifen treated Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/+ control chimeras (right panel). Isotype control sample is shown for both sets of analysis. (b, c) Flow cytometry analysis showing the proportion of p27+ MZ B cells in Zfp36l1fl/flMb1+/+ and Zfp36l1fl/flMb1Cre/+ mice (b), tamoxifen treated Zfp36l1+/+R26ERT2-Cre/+ and Zfp36l1fl/flR26ERT2-Cre/+ chimeric mice (c). (d) Flow cytometry analysis showing Ki67 staining in MZ, Fo and GC B cells from Zfp36l1fl/flMb1Cre/+ mice and Zfp36l1fl/flMb1+/+ littermate controls. FMO control sample is shown for all sets of analysis. (e) Proportion of Ki67+ cells in Zfp36l1fl/flMb1Cre/+ and Zfp36l1fl/flMb1+/+. (g) DAPI staining in MZ and Fo B cells from Zfp36l1fl/flMb1Cre/+ mice and Zfp36l1fl/flMb1+/+ littermate controls. Numbers on the graph indicate the percentage of cells in the gate. (h, i) Proportion of cells in the S/G2-M phase of the cell cycle in MZ (h) and Fo B cells (i). Sample sizes: b (Zfp36l1fl/flMb1+/+ 9, Zfp36l1fl/flMb1cre/+ 7), c (Zfp36l1+/+R26ERT2-Cre/+ 6, Zfp36l1fl/flR26ERT2-Cre/+ 8), e, h, i (5) *P ≤ 0.05, Mann-Whitney. Data pooled from 2 independent experiments (b; mean), data from 1 experiment (c, e, h, i; mean).

Supplementary Figure 7 ZFP36L1 regulates localization, identity and survival.

(a) Immunofluorescence analysis of spleen sections from Zfp36l1fl/flR26ERT2-Cre/+ mice and R26GFPZFP36L1Cd23-Cre+ mice. Sections were stained with anti-CD1d (red) and anti-IgD (green). Images were captured at 20x magnification, scale bars represent 50μm. White arrows on the R26GFPZFP36L1Cd23-Cre+ image indicate colocalization of the IgD and CD1d, white arrows on the Zfp36l1fl/flR26ERT2-Cre/+ image indicate single stained cells only. (b) Day 4 NP-specific IgM endpoint titres from mice immunized with NP-α-GalCer. (c) Schematic model depicting the mechanism by which ZFP36L1 regulates survival, migration and identity of MZ B cells through a network of transcription factors which include KLF2 and IRF8. Sample sizes: b (3), Mann-Whitney. Data pooled from 1 experiment (b; mean).

Supplementary Figure 8 ZFP36L1 controls genes that encode products involved in the trafficking of mature B cells.

(a) Venn diagram indicating the number of overlapping genes, which are increased in Zfp36l1fl/flR26ERT2-Cre/+ (KO) MZ B cells when compared to Zfp36l1+/+R26ERT2-Cre/ERT2-Cre (WT) MZ B cells, and decreased in R26GFPZFP36L1Cd23-Cre+ (GFP) Fo when compared to R26GFPZFP36L1Cd23-Cre- (WT) B cells using a padj<0.05 cutoff. (b) Pathway analysis of genes which are increased in expression in KO MZ B cells and decreased in expression in GFP expressing Fo B cells as defined in a using a padj<0.05 cutoff. (c, d) S1pr1 mRNA expression in Zfp36l1fl/flR26ERT2-Cre/+ MZ B cells and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre control MZ B cells (c), and R26GFPZFP36L1Cd23-Cre+ and R26GFPZFP36L1Cd23-Cre- Fo B cells (d). (e) Follicular area in image analysis was defined using CD169 (MOMA-1) staining (green). Follicular area is shown by yellow line. MFI was then calculated for this area. MZ area was defined as CD1d+ (magenta) area outside MOMA-1 (green) and IgD (not shown) staining. MZ area is shown by white line. Background fluorescence (grey box) was calculated and subtracted from MFI. Sample sizes: c, d (4), Benjamini Hochburg adjusted p values calculated using DESeq2. Data from 1 experiment (c, d; mean + s.e.m.).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 7–9 (PDF 1735 kb)

Supplementary Table 1

Differentially expressed genes in MZ B cells from Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre mice. Differentially expressed genes (P adj < 0.01) in comparisons of MZ B cells from tamoxifen-treated Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre mice. A 1.5-fold change cutoff has been applied. (XLSX 62 kb)

Supplementary Table 2

Upregulated genes (P adj < 0.01) for GSEA showing iCLIP hits and AREs. Genes that are increased in expression (P adj < 0.01) in comparisons of MZ B cells from tamoxifen-treated Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre mice. Hits in iCLIP analysis and ARE found in the 3′ UTR of the gene are indicated. An FDR of 0.05 was used for the iCLIP data. (XLSX 87 kb)

Supplementary Table 3

Downregulated genes (P adj < 0.01) for GSEA showing iCLIP hits and AREs. Genes that are decreased in expression (P adj < 0.01) in comparisons of MZ B cells from tamoxifen-treated Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre mice. Hits in iCLIP analysis and ARE found in the 3′ UTR of the gene are indicated. An FDR of 0.05 was used for the iCLIP data. (XLSX 75 kb)

Supplementary Table 4

Differentially expressed genes in MZ B cells from Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre mice compared to wild-type MZ and Fo B cells. Differentially expressed genes (P adj < 0.01, and subject to a 1.5-fold cutoff) in comparisons of Zfp36l1fl/flR26ERT2-Cre/+ (KO) MZ B cells and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre control (WT) MZ B cells to wild-type Fo B cell gene expression. Gene lists have been separated into each quadrant of the correlation dot plot. Zfp36l1 has been removed from this analysis. (XLSX 65 kb)

Supplementary Table 5

Differentially expressed genes in Fo B cells from R26GFPZFP36L1Cd23-Cre+ and R26GFPZFP36L1Cd23-Cre–versus wild-type MZ and Fo B cells. Differentially expressed genes (P adj < 0.01, and subject to a 1.5-fold cutoff) in comparisons of R26GFPZFP36L1Cd23-Cre+ (GFP) Fo B cells and R26GFPZFP36L1Cd23-Cre– control Fo B cells to wild-type MZ B cell gene expression. Gene lists have been separated into each quadrant of the correlation dot plot. Zfp36l1 has been removed from this analysis. (XLSX 57 kb)

Supplementary Table 6

Differentially expressed genes in comparisons of MZ B cells from Zfp36l1fl/flR26ERT2-Cre/+ and Zfp36l1+/+R26ERT2-Cre/ERT2-Cre mice to Fo B cells from R26GFPZFP36L1Cd23-Cre+ and R26GFPZFP36L1Cd23-Cre– mice. Differentially expressed genes (P adj < 0.05) found in RNA-seq analysis comparing MZ B cells from Zfp36l1fl/flR26ERT2-Cre/+ to Zfp36l1+/+R26ERT2-Cre/ERT2-Cre controls and RNA-seq analysis comparing GFP–ZFP36L1-positive Fo B cells to Fo B cells from littermate controls. Gene lists have been separated into each quadrant of the correlation dot plot. Zfp36l1 has been removed from this analysis. (XLSX 77 kb)

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Newman, R., Ahlfors, H., Saveliev, A. et al. Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1. Nat Immunol 18, 683–693 (2017). https://doi.org/10.1038/ni.3724

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