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Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation

Nature Immunology volume 17, pages 1216–1225 (2016)Cite this article

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Abstract

The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

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Figure 1: B cell differentiation is coupled to unique transcriptional states.
Figure 2: B220intCD138+ plasmablasts and B220loCD138+ plasma cells undergo DNA hypomethylation.
Figure 3: DML are 'preferentially' present at B cell enhancers and near motifs of transcription factors required for B cell differentiation.
Figure 4: Inhibition of DNA methylation facilitates plasma cell differentiation.
Figure 5: Transcriptional amplification and DNA hypomethylation coincide with cellular division.
Figure 6: Cell-division-coupled changes in gene expression and DNA methylation.
Figure 7: Dynamic gene-expression changes correspond with a hierarchy of DNA hypomethylation.
Figure 8: Gene expression correlates with cell division and DNA methylation.

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Acknowledgements

We thank R. Martinez for flow cytometry; R. Butler for mouse care; P.M. Vertino, P.A. Wade, L.H. Boise and H.D. Kondilis-Mangum for comments and critique in reading the manuscript; K.N. Conneely and H. Wu for statistical advice; the Genome Technology Center at NYU for Illumina sequencing; the Emory Flow Cytometry Core for flow cytometry; and the Emory Integrated Genomics Core for high-sensitivity DNA Bioanalyzer analysis. Supported by Emory University School of Medicine (institutional funds to J.M.B.) and the US National Institutes of Health (R01 GM47310, R01 AI123733 and U19 AI110483 to J.M.B.; F31 AI112261 to B.G.B.; T32 GM008490 to J.M.B. and B.G.B.; and T32 AI007610 to A.P.R.B.).

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  1. Benjamin G Barwick and Christopher D Scharer: These authors contributed equally to this work.

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  1. Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, Georgia, USA

    Benjamin G Barwick, Christopher D Scharer, Alexander P R Bally & Jeremy M Boss

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Contributions

B.G.B. contributed to experiment conception and design, performed the DNA-methylation analyses, mouse experiments, RNA-seq analysis and bioinformatics analyses and wrote the paper; C.D.S. contributed to experimental conception and design and performed RNA microarray analysis; A.P.R.B. provided technical expertise to mouse experiments; J.M.B. contributed to experimental conception and design and wrote the paper; and all authors provided editorial input.

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Integrated supplementary information

Supplementary Figure 1 LPS induces a robust B cell response.

Comparison of spleen (a) size, (b) weight and (c) total splenocytes from naïve and day 3 LPS challenged mice. (d) Representative analysis of splenic B cell frequency in naïve and LPS-challenged mice indicate proportional increase in B cell frequency primarily of the GL7+ activated population, with quantitation of (e) total B220+ B cells and (f) GL7+ activated B cells per spleen. *P <0.001, Welch’s t-test. Data are from two independent experiments with 7 and 8 mice per experiment (b, c, e, f; mean and s.d).

Supplementary Figure 2 Validation of statistically representative and biologically relevant differentially methylated loci (DML).

(a, d, g, j, m) RRBS data are shown for B220+ B cells (B; blue), B220intCD138+ plasmablasts (PB; burgundy), and B220loCD138+ plasma cells (PC; gold) for DML near Irf4, Il10, Tnfrsf13b, Tnfrsf13c, and Egr3. (b, e, h, k, n) Genome schematic of regions near the validated DML. RRBS coverage is shown with vertical black lines and the DML interrogated is indicated with a black arrow. Primer regions are shown below coverage. UCSC KnownGenes are plotted with exonic and intronic sequence denoted with thick and thin black lines, respectively. Scale on the bottom right indicates 1 kb. (c, f, i, l, o) combined bisulfite restriction analysis validation of DML. Enzyme digested and mock digested samples are run adjacent on the agarose gel and indicated by a “+” and “-“ at the bottom of the gel image, respectively. Biological replicates are indicated on top. B220intCD138+ Plasmablasts and B220loCD138+ plasma cells were obtained from the same mice. Six mice were used in this experiment.

Supplementary Figure 3 DNA methylation in B cell subsets.

Plasmablast and plasma cell DML near Cd86, Irf4, Prdm1, and Il10 were validated in naïve B cells (nB), GL7+ activated B cells (aB), B220intCD138+ splenic plasmablasts (PB), B220loCD138+ splenic plasma cells (PC), B220intCD138+ bone marrow cells (BMPB), and B220loCD138+ bone marrow cells (BMPC). DNA methylation was measured as a ratio of mock digested DNA to HpaII digested DNA using qPCR primers. MspI digested DNA serves as a negative control. The experiment was performed once with 4 biological replicates.

Supplementary Figure 4 Tracking of cell division in an in vivo model B cell differentiation.

(a) Schematic of experimental design. (b) Flow cytometry showing CD45.1+ B cells in the spleen of μMT hosts 3 days post-LPS challenge. (c) CFSE staining on CD45.1+ cells from LPS challenged and control mice. (d) Quantification of divisions for LPS and control mice. (e) Flow cytometry analysis of CFSE and viability exclusion dye on CD45.1+ cells transferred into μMT hosts that were challenged with LPS as described in Fig 5. (f) CFSE and B220 expression showing B220+ and B220− populations. (g) Histogram of viability exclusion dye on B220+ and B220− populations. (h) Histogram of CD138 expression on B220+ and B220− populations. Data are from two experiments with 4 and 6 mice per experiment (a-d) or one experiment with 6 mice (e-h) (d; mean and s.d.).

Supplementary Figure 5 Validation of division-specific DNA-methylation and gene-expression changes.

(a) Flow cytometry analysis (top) and post-sort purity (bottom) of transferred splenic CD45.1+B220+ B cells in μMT hosts 3 days post-LPS challenge. (b) Schematic of differentially expressed genes with proximal differentially methylated loci that were interrogated for division-linked gene expression and DNA methylation changes. The interrogated CpG loci are denoted by the black arrows. (c) Gene expression for the genes shown in b and the populations in a. Expression was determined using RT-qPCR and is plotted as % 18S. (d) DNA methylation for the CpG loci denoted in b. DNA methylation was determined using qPCR on HpaII digested DNA relative to mock digested DNA. The methyl-insensitive enzyme MspI was used as a negative control. (e) Flow cytometry analysis as in a, that was used to analyze specific cell divisions using RNA-seq and RRBS. Data is from Fig 5e reproduced here for clarity. (f) Gene expression determined using RNA-seq for the genes in b and the populations in e. (g) DNA methylation determined using RRBS for the CpG loci shown in b and the populations in e. Data are from two with 6 and 4 mice (a), one experiment with 3 mice (c-d) or.one experiment with 2 mice (f-g) (b, d, f, g; mean and s.d.).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 943 kb)

Supplementary Table 1

S1-DEGs.v2.xlsx (XLSX 8983 kb)

Supplementary Table 2

S2-DEG.Gene.Ontology.v2.xlsx (XLSX 1128 kb)

Supplementary Table 3

S3-DEG.GSEA.v3.xlsx (XLSX 1871 kb)

Supplementary Table 4

S4-SequencingStats.v3.xlsx (XLSX 13 kb)

Supplementary Table 5

S5-DML.v3.xlsx (XLSX 59643 kb)

Supplementary Table 6

S6-MetaAnalysis.v2.xlsx (XLSX 13 kb)

Supplementary Table 7

S7-Primers.v1.xlsx (XLSX 16 kb)

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Barwick, B., Scharer, C., Bally, A. et al. Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation. Nat Immunol 17, 1216–1225 (2016). https://doi.org/10.1038/ni.3519

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