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Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement

Nature Immunology volume 4, pages 124–131 (2003)Cite this article

Abstract

Polycomb group protein Ezh2 is an essential epigenetic regulator of embryonic development in mice, but its role in the adult organism is unknown. High expression of Ezh2 in developing murine lymphocytes suggests Ezh2 involvement in lymphopoiesis. Using Cre-mediated conditional mutagenesis, we demonstrated a critical role for Ezh2 in early B cell development and rearrangement of the immunoglobulin heavy chain gene (Igh). We also revealed Ezh2 as a key regulator of histone H3 methylation in early B cell progenitors. Our data suggest Ezh2-dependent histone H3 methylation as a novel regulatory mechanism controlling Igh rearrangement during early murine B cell development.

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Figure 1: Expression of Ezh2 and Ezh1 mRNA in B lineage cells.
Figure 2: Conditional inactivation of Ezh2.
Figure 3: Impaired development of Ezh2-deficient B cells.
Figure 4: Expression of transgenic BCR rescues the development of Ezh2-deficient B lineage cells.
Figure 5: Impaired rearrangement and expression of VHJ558 family genes in Ezh2-deficient pro-B cells.
Figure 6: Ezh2 does not control expression of IgH germline transcripts, IL-7–mediated STAT5 activation or histone acetylation in pro-B cells.
Figure 7: Reduced lysine methylation of histone H3 in Ezh2-deficient pro-B cells.

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Acknowledgements

We thank M. Nussenzweig, K. Rajewsky, C. Schmedt, K. Saijo, I. Mecklenbräuker and D. O′Carroll for discussions. We also thank G. Hannon for critical review of this manuscript. Supported by The Irene Diamond Fund (A.T.), National Institutes of Health grant (A.T.), NIH, RR0086 (B.T.C.) and The Rockefeller University's Norman and Rosita Winston Fellowship Program (I.S.).

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Authors and Affiliations

  1. Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, 10021, NY, USA

    I-hsin Su, Ashwin Basavaraj & Alexander Tarakhovsky

  2. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, 10021, NY, USA

    Andrew N. Krutchinsky & Brian T. Chait

  3. Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, New York, 10032, NY, USA

    Oliver Hobert

  4. Department of Molecular Biology, Max Planck Institute for Biochemistry, D-82152, Martinsried, Germany

    Axel Ullrich

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Supplementary information

Supplementary Fig. 1.

Efficiency of Mx-Cre–mediated Ezh2 deletion in bone marrow B lineage populations. The efficiency of the Cre-mediated Ezh2 deletion was quantified by densitometry analysis of Southern blots (a), PCR (b) and RT-PCR (c). (PDF 833 kb)

Supplementary Fig. 2.

Ezh2-deficient B lineage cell development in the bone marrow chimeric mice. Bone marrow cells isolated from poly(I)·poly(C) treated Ezh2fl/fl Mx-Cre or control Ezh2fl/fl mice were transferred into lethally irradiated C57BL/6 mice. The histogram shows the distribution of Ly9.1+ and Ly9.1– cells in the bone marrow of the chimera mice. The bars indicate the gate used for the analysis of Ly9.1+ cells (top panel). The Ly9.1+ donor derived cells were analyzed for the expression of surface IgM and B220 (middle panel). The surface IgM negative cells were gated and analyzed for their expression of CD43 and B220. The numbers indicate the percentages of gated cells. (PDF 1212 kb)

Supplementary Fig. 3.

The expression of the κ light chain in pro-B cells is not affected by Ezh2 deficiency. Intracellular κ chain expressing cells were identified using κ chain-specific antibody (R33-18-10). The numbers indicate the percentages of κ chain positive cells within the pro-B cell population. (PDF 994 kb)

Supplementary Fig. 4.

Ezh2 does not control peripheral B cell maturation in vivo and activation in vitro. (a) Bone marrow cells were analyzed for expression of indicated surface proteins. (b)The Ezh2fl/fl CD19-Cre pro-B, pre-B and splenic B cells were purified by FACSort and expression levels of mRNAs were analyzed by RT-PCR (lane 1: pro-B cells, lane 2: pre B cells, lane 3: splenic B cells, lane 4: Ezh2–/– BM cells, lane 5: Ezh2fl/fl cells, lane 6: ladder). (c) B cells were isolated from splenic cell suspension by MACS-depletion of non-B CD43+ cells. Purified B cells were labeled with CFSE, incubated with different stimuli for 4 days and cell proliferation was measured by FACS. Histogram shows the CFSE fluorescence levels with the filled area showing the fluorescence of untreated cells. (d) Splenocytes were loaded with Ca2+ sensitive dye Indo-1 followed by incubation with the PE-labeled anti-B220 antibody. Calcium mobilization was initiated by anti-IgM antibody stimulation (indicated by an arrow) and measured as described before49. (PDF 1804 kb)

Supplementary Fig. 5.

Ezh2-deficient pro-B cells are equipped to carry V(D)J rearrangement. (a) Expression of mRNAs, encoding proteins essential for the V(D)J rearrangement is not altered in the absence of Ezh2. Total RNA was isolated from control and Ezh2–/– pro-B cells. The expression of Rag-2, DNA-PK and Ku-80 was analyzed by RT-PCR. The expression of HPRT was used to confirm equal amount of template in the samples. (b) DNA breaks of VHJ558 signal and coding ends were analyzed by LM-PCR. Primers specific for VHJ558 genes were used to amplify coding end. The signal end was visualized with primers annealing downstream from recombination signal sequences (RSSs) of many VH558 family genes. Arrows indicate the positions of DSBs. Rag-2 deficient mice were used as negative control. (PDF 775 kb)

Supplementary Fig. 6.

Ezh2-deficient pro-B cells are viable and cycling. (a) The frequency of apoptotic cells within the CD43+B220+ pro-B cell population was analyzed by TUNEL (upper panel) or by intracellular staining of activated caspases (lower panel). The numbers indicate the percentages of TUNEL positive cells and activated caspase-positive dead cells (upper right quadrant) or caspase-positive live cells (lower right quadrant). (b) BrdU labeling of control and Ezh2 deficient pro-B cells. Mice received 200 mg of BrdU by intraperitoneal (i.p.) injection and were sacrificed at different time points (1, 2 and 4 hours) after injection. The incorporated BrdU was detected by intracellular staining with anti-BrdU antibody. The DNA content was analyzed with 7AAD. (PDF 3473 kb)

Supplementary Fig. 7.

Expression of IL-7 receptor is not affected by Ezh2 deficiency. Bone marrow cells isolated from poly(I)·poly(C) treated Ezh2fl/fl Mx-Cre or control Ezh2fl/fl mice were incubated with anti-B220, anti-CD43 and anti-IL-7Rα antibodies and analyzed by FACS. The histograms show the expression levels of surface IL-7Rα (gray area). The thick line indicates the fluorescence of cells incubated with the isotype-matched control antibody. The numbers indicate the mean value of the fluorescence. (PDF 671 kb)

Supplementary Fig. 8.

Reduced lysine methylation of histone H3 in Ezh2-deficient pro-B cells. Methylation of histone H3 in nuclear lysates of pro-B cells incubated in the absence or presence of IL-7 was analyzed by immunoblotting using anti-pan–methyl-lysine (a,b) or dimethyl-histone H3-K9– or H3-K4–specific antibodies (a). The amount of histone H3 was controlled by immunoblotting with anti-histone H3 antibodies. The numbers indicate fold change compared to the signal of unstimulated control lysate after normalized against the amount of histone H3 (a). Three independent experiments are shown. (PDF 907 kb)

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Su, Ih., Basavaraj, A., Krutchinsky, A. et al. Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nat Immunol 4, 124–131 (2003). https://doi.org/10.1038/ni876

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