Abstract
Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.
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Acknowledgements
The authors are grateful to S. Richard (McGill University) and to K. Rajewsky (Max Delbrück Center) for providing the Prmt1f/f and CD19-cre mouse lines, respectively. The authors thank C. Sainz-Rueda, B. Knapp, S. Welz and Y. Kori for their technical assistance, and S. Herzog (Biocenter Innsbruck) and M. Jung, (Institute of Pharmaceutical Sciences) for providing reagents. Furthermore, we thank P.J. Nielsen, L. Leclercq and E. Molnár for critically reading the manuscript. Work in the laboratory of M.R and B.W. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; TRR130) and the Excellence Initiative of the German Federal & State Governments (Grant EXC 294 BIOSS Centre for Biological Signalling Studies). The work in the laboratory of M.R. was also supported by ERC-grant 32297, the German Cancer Foundation grant 111026 and by the Deutsche Forschungsgemeinschaft through SFB746. G.J.F. is financially supported by the DFG through MI1942/2-1 to S.M. D.M.T. is supported by a National Health and Medical Research Council (NHMRC) Australia program grant (1054925), S.I. by a DRFG Fellowship and D.M.T. by an NHMRC Research Fellowship.
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E.D. designed and performed experiments, analyzed data and wrote the manuscript. S.I. designed and performed experiments, and analyzed data. F.D. performed experiments and analyzed data. T.B., A.S., T.W., G.J., andS.M. performed experiments. B.W. designed experiments and analyzed data. D.T. designed experiments and analyzed data. H.J. provided cells, reagents, and helped with experimental design and data interpretation. D.M. conceived the study, performed experiments, analyzed data and wrote the manuscript. M.R. conceived the study, analyzed data and wrote the manuscript.
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Supplementary Figure 1 PRMT1-deficiency impairs B cell development
(a) Confirming the deletion of the Prmt1 floxed allele. Splenic B cells from Prmt1+/+-CD19+/cre (samples: 1 and 4), Prmt1fl/fl-CD19+/cre (samples 2 and 5), tail biopsy of a Prmt1fl/+-CD19+/cre mice (sample 3) or from a heterozygous knock out, generated by germline Cre activity, (sample 6) were isolated using a B cell isolation kit and LS magnetic column (Miltenyi Biotech). B cell purity (>98%) was determined on the FACS CantoII (BD) with CD19 and B220 antibodies (data not shown). Genotyping PCR was performed as previously described46. (b,c) Analysis of bone marrow chimeras that were generated by injecting mice with a mixture of 50% Ly5.1+ wild type and 50% of Ly5.2+ Prmt1fl/flRosa26+/+ (Group 1) or 50% Ly5.1+ wild type and 50% of Ly5.2+ Prmt1fl/flRosa26ERT2Cre/+ (Group 2) bone marrow cells. Four weeks after reconstitution the mice received 3 doses of Tamoxifen to induce Prmt1 deletion and were then sacrificed and analyzed one week after the last injection. Expression of the indicated surface markers was detected by flow cytometry. The numbers represent percentages of cells in the indicated gates. The data shown are representative of five mice with similar results. Statistical analysis of the data depicted in (b) was performed using the Mann–Whitney test (**, P < 0.01; n = 5. The line shows the median). Statistical analysis of data depicted in (c) was performed using the Student's t-test (**, P < 0.01; n = 4 or 3 respectively. The line shows the mean) (d) Flow cytometry analysis of surface κLC expression of freshly isolated wild type CD19+ κLC− bone marrow cells retrovirally transduced with vectors (IRES-GFP) encoding either non-targeting shRNA (shControl) or Prmt1-targeting shRNA (shPRMT1) and cultured for 72 h with or without IL-7. The percentages of κLC positive cells among the transduced (GFP+) pre-B cells are indicated. Data shown are representative of three independent experiments with similar results. The values of the κLC+ cells in the experimental samples were normalized to the corresponding control sample and the statistical analysis was performed using the two-tailed Student's t-test (depicted as mean ± SD n=3 independent experiments)
Supplementary Figure 2 BTG2 promotes pre-B cell differentiation
(a) 1676 cells were transduced with a retroviral vector (IRES-GFP) encoding BTG2, BTG2ΔBoxC or an empty control vector (EV). Cells were cultured in medium with IL-7 and the proportion of κLC surface-expressing cells was measured 72h post transduction by flow cytometry. Percentages of κLC positive cells are indicated for transduced (GFP+) cells. The numbers show the mean ± SD. Statistical analysis, comparing EV to BTG2 or BTG2ΔBoxC, was performed using the two-tailed Student's t-test (n=3 independent experiments). (b) Flow cytometry analysis of surface κLC expression of freshly isolated wild type CD19+ κLC− bone marrow cells retrovirally transduced with vectors (IRES-GFP) encoding an empty control vector (EV), BTG2 or BTG2ΔBoxC and cultured for 72 h with or without IL-7. The percentages of κLC+ cells among the transduced (GFP+) pre-B cells are indicated. The numbers show the mean ± SD. Statistical analysis was performed using the two-tailed Student's t-test comparing EV to BTG2 or BTG2ΔBoxC. The values of the experimental samples were normalized to the corresponding control sample and the statistical analysis was performed using the two-tailed Student's t-test (depicted as mean ± SD n=3 independent experiments) (c) Flow cytometry analysis of surface κLC expression of freshly isolated wild type CD19+ κLC− bone marrow cells retrovirally transduced with vectors (IRES-GFP) encoding either non-targeting shRNA (shControl) or BTG2-targeting shRNA (shBTG2) and cultured for 72 h with or without IL-7. The percentages of κLC positive cells among the transduced (GFP+) pre-B cells are indicated. The values of the experimental samples were normalized to the corresponding control sample and the statistical analysis was performed using the two-tailed Student's t-test (depicted as mean ± SD n=3 independent experiments) (d) Western blot analysis of total cellular lysates of 1676 pre-B cells transduced with a retroviral vector (IRES-GFP) encoding BTG2, BTG2ΔBoxC or empty control vector (EV) and blotted with anti-asymmetric dimethyl-arginine antibody (ADMA). Actin was used as a loading control. Data are representative of two independent experiments with similar results. (e) Freshly isolated wild type CD19+ κLC− bone marrow cells were transduced with retroviral vectors (IRES-GFP) encoding either BTG2, BTG2ΔBoxC or an empty control vector (EV) and cultured without sorting. The ratio of GFP+/GFP− cells was measured over time by flow cytometry. The percentage of GFP+ cells was normalized to the value of the measurement starting point of the respective sample. Data are shown as mean ± SD. Statistical analysis was performed using the two-tailed Student's t-test (***, P < 0.001; n=3 independent experiments).
Supplementary Figure 3 Schematic model showing the suggested mechanism of the pre-BCR signaling leading to the methylation-dependent disruption of the CDK4-Cyclin-D3 complex.
In proliferating pre-B cells PI(3)K-Akt signaling inhibits Foxo1. In parallel, BTG2 is weakly expressed and the CDK4–Cyclin D3 complex drives cell-cycle. During differentiation, Foxo1 is stabilized and upregulates BTG2, that in turn activates PRMT1 to methylate CDK4. Methylated CDK4 dissociates from Cyclin D3, leading to its degradation. This causes a cell-cycle block, allowing the expression of RAG1, the stabilization of RAG2, and subsequently the rearrangement of the immunoglobulin light chain gene.
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Dolezal, E., Infantino, S., Drepper, F. et al. The BTG2-PRMT1 module limits pre-B cell expansion by regulating the CDK4-Cyclin-D3 complex. Nat Immunol 18, 911–920 (2017). https://doi.org/10.1038/ni.3774
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DOI: https://doi.org/10.1038/ni.3774
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