BTG2–PRMT1 Protein Complexes Antagonize Pre-B-Cell Proliferation to Promote B-Cell Development

RESEARCH HIGHLIGHT

Flip the switch: BTG2–PRMT1 protein complexes antagonize pre-B-cell proliferation to promote B-cell development

Glendon S Wu1,2 and Craig H Bassing1,2

Cellular & Molecular Immunology advance online publication, 12 February 2018; doi:10.1038/cmi.2017.156

B lymphocytes are vital components of Rag2 and activate transcription of Cyclin primary pre-B cells to determine the vertebrate adaptive immune systems. D3 (Ccnd3). Cyclin D3 binds to and rolesofBTG2andPRMT1indeveloping However, B-cell dysfunction can pro- activates cyclin-dependent kinase 4 B cells. They found that B-lineage-spe- mote autoimmunity and immunodefi- (CDK4) to drive G1-phase cells into cificdeletionofPrmt1 resulted in a ciency, and errors in development can the cell cycle (Figure 1a). Although IL-7 defect in early B-cell development as cause B-lineage lymphoid malignancies. antagonizes Ig gene assembly and pro- reflected by lower numbers of cells at B-cell development requires RAG1/ motes pre-B-cell proliferation, pre-BCR the pro-B-cell stage onwards. Using a RAG2 (RAG) endonuclease-mediated signaling in pre-B cells has opposing pre-B-cell line (1676) and ex vivo cul- assembly of immunoglobulin (Ig) genes. effects by stimulating Rag1/Rag2 tran- tured primary pre-B cells, knockdown of The assembly of Ig heavy chain (IgH) scription and repressing Ccnd3 transcrip- Prmt1 mRNA in non-cycling cells genes in G1-phase pro-B cells produces tion. As pre-B cells lose contact with the resulted in a reduction of Igκ+ B cells, IgH proteins that pair with VpreB/λ5to IL-7 niche, pre-BCR signaling triggers indicating that PRMT1 regulates pre-B- form pre-B-cell receptor (pre-BCR) G1 arrest and the assembly of Ig light cell differentiation. Curiously, overex- complexes. In pro-B cells, pre-BCR sig- chain (IgL) genes (Igκ or Igλ) pression of Prmt1 did not enhance pre- naling inhibits Rag1/Rag2 transcription, (Figure 1b). In this research highlight, B-cell development, suggesting other cel- stabilizes Cyclin D3 to drive cellular we discuss recent work from Dolezal lular factors limit PRMT1 activity. Given proliferation and promotes differentia- et al.1 that uncovers an important role that PRMT1 and BTG2 complex tion of large pre-B cells. Interleukin (IL)- for B-cell translocation gene (BTG2) and in vitro,3 the authors quantified the 7 augments this developmental transition protein arginine methyl transferase 1 BTG2 expression in developing B cells. by signaling through the IL-7 receptor (PRMT1) in posttranslational inactiva- They found that non-cycling 1676 pre-B (IL-7R) to inhibit transcription of Rag1/ tion of Cyclin D3/CDK4 complexes. This cells exhibited elevated levels of Btg2 mechanism suppresses pre-B-cell prolif- transcript and protein relative to cycling eration and activates IgL gene assembly cells, mirroring the expression pattern of 1 Immunology Graduate Group, Children’s Hospi- and pre-B-cell differentiation.1 Prmt1. BTG2 contains a BoxC region tal of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, BTG2 is an antiproliferative BTG/ that is required for BTG2 to bind and 5 PA 19104, USA and 2Divsion of Cancer Patho- transducer of ErbB2 family member that activate PRMT1. Overexpression of biology, Department of Pathology and Laboratory inhibits the G1/S transition of the cell wild-type BTG2, but not a BTG2 mutant ’ Medicine, Children s Hospital of Philadelphia, cycle.2 BTG2 binds and activates with a BoxC deletion (BTG2ΔBoxC), Abramson Family Cancer Research Institute, κ Perelman School of Medicine at the University PRMT1, forming complexes that methy- enhanced Ig expression and pre-B-cell of Pennsylvania, Philadelphia, PA 19104, USA late protein substrates.3 Prior experi- differentiation in 1676 and ex vivo cul- Correspondence: Dr CH Bassing, PhD, Immunol- ments suggested that BTG2 and tured pre-B cells. These effects were ’ ogy Graduate Group, Children sHospitalofPhi- PRMT1 each had a role in promoting dependent on PRMT1 as PRMT1 knock- ladelphia, Perelman School of Medicine at the 4 University of Pennsylvania, 4054 Colket Transla- B-cell development; however, the down, even in the presence of over- tional Research Building, 3510 Civic Center mechanisms by which these two proteins expressedBTG2,reducedIgκ Boulevard, Philadelphia, PA 19104, USA. regulated B-cell differentiation were not expression. BTG2 knockdown in 1676 E-mail: [email protected] explored. Dolezal et al.1 conducted and ex vivo cultured pre-B cells also Received: 16 November 2017; Accepted: 25 κ+ November 2017 experiments in mice and in cultured reduced the frequency of Ig cells. proliferation to promote B-cell development GlendonS Wu and CraigH Bassing

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Figure 1 Signals derived from the pre-B-cell receptor (pre-BCR) and from the interleukin-7 receptor (IL-7R) are required for the cellular survival, proliferation and differentiation of developing B cells. IL-7 is produced by bone marrow stromal cells and induces pre-B-cell proliferation. (a) IL-7R signaling activates a signaling network that promotes the transcription of Ccnd3 while inhibiting Rag1/Rag2 and Btg2 transcription. Cyclin D3 in complex with in cyclin-dependent kinase 4 (CDK4) stimulates S-phase entry and RAG2 degradation. (b) After escape from the IL-7 niche, pre-BCR signaling promotes G1 cell cycle arrest and Rag1/Rag2 and Btg2 transcription and antagonizes Ccnd3 transcription. B-cell translocation gene (BTG2) complexes with protein arginine methyl transferase 1 (PRMT1), leading to methylation of arginine residues CDK4. CDK4 methylation disrupts its association with Cyclin D3, thus inhibiting cellular proliferation and promoting immunoglobulin light chain recombination and pre-B-cell differentiation.

PRMT1 functions to asymmetrically Cyclin D3 expression. Indeed, overex- Dolezal et al.1 next assessed whether dimethylate arginine residues (ADMA).6 pression of wild-type BTG2, but not the Cyclin D3 or CDK4 were targets of Consistent with the cellular phenotype BTG2ΔBoxC mutant, resulted in a dras- PRMT1-mediated methylation. They data, analysis of ADMA revealed exten- tic reduction in Cyclin D3 protein in a found that CDK4, but not Cyclin D3, sive methylation in cells transduced with proteasome-dependent manner. Conver- was specifically methylated by PRMT1 wild-type BTG2 but not the BTG2ΔBoxC sely, BTG2 or PRMT1 knockdown using a fluorescent in vitro methylation mutant. Collectively, these data indicate increased Cyclin D3 protein and drove assay. Moreover, CDK4 incubated with that BTG2 and PRMT1 function together cell cycle progression. These data suggest active PRMT1 and S-adenosyl methio- to promote pre-B-cell development. that BTG2 and PRMT1 cooperate to nine failed to bind Cyclin D3, indicating Ig gene assembly occurs in G1-phase arrest pre-B cells in G1 by licensing that Cyclin D3/CDK4 complexes are cells and requires the expression of both Cyclin D3 degradation. As Cyclin D3 unstable when CDK4 is methylated by RAG1 and RAG2 proteins.7 Thus the fuels cell cycle progression, the authors PRMT1. Analysis of CDK4 methylation authors next tested the effect of BTG2 on hypothesized that Cyclin D3 is an inhi- by mass spectrometry revealed seven RAG1 and RAG2 expression in develop- bitor of the pre-B-cell differentiation arginine residues that are targets of ing pre-B cells. 1676 pre-B cells trans- program. Indeed, 1676 pre-B cells trans- PRMT1, and of these, four residues duced with wild-type BTG2 exhibited an duced with Cyclin D3 exhibited a (R55, R73, R82 and R163) were posi- increase in RAG2 protein as compared decrease in the frequency of Igκ+ B cells. tioned at the surface where Cyclin D3 with control and BTG2ΔBoxC mutant Conversely, knockdown of Cyclin D3 binds. To test whether these residues are cells. As RAG2 is degraded in S phase,8 increased the frequency of Igκ+ 1676 required for PRMT1 to disrupt the Dolezal et al.1 reasoned that the increase pre-B cells. Notably, combined overex- Cyclin D3/CDK4 complex, the authors in RAG2 protein in BTG2-transduced pression of Cyclin D3 and BTG2 led to a mutated all four arginines to lysine cells was due to G1 cell cycle arrest. defect in the development of Igκ+ B cells, (CDK44R-K) and subjected the mutant Consistent with this notion, the authors indicating that Cyclin D3 is a potent protein to the fluorescent methylation found that 1676 pre-B cells expressing inhibitor of pre-B-cell differentiation. and co-immunoprecipitation assays. As wild-type BTG2 had a higher fraction of Collectively, these data reveal a critical expected, the CDK44R-K mutant was cells arrested in G1 phase. Given that role for the BTG2–PRMT1 complex in refractory to PRMT1-mediated methyla- IL-7R signaling promotes Cyclin D3 promoting Cyclin D3 degradation to tion compared with wild-type CDK4, expression to drive cells through G1 suppress cell cycle progression, stimulate resulting in its stable association with and into S phase,9 the authors asked RAG expression and thereby promote Cyclin D3. Presumably, the increased whether BTG2 and PRMT1 reduce pre-B-cell development. stability of Cyclin D3/CDK44R-K

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complexes would promote cell cycle detected in the bone marrow or spleen, and PRMT1. The BTG2/PRMT1 axis progression. To test this idea, the authors indicating that in vitro and in vivo BTG2 highlights a unique posttranslational overexpressed the CDK44R-K mutant in and PRMT1 restricts the proliferation of mechanism that regulates pre-B-cell pro- 1676 pre-B cells and measured growth. BCR-ABL1-transformed pre-B cells. liferation and differentiation. This Indeed, the cells transduced with the This study advances our understand- mechanism may have important impli- CDK44R-K mutant expanded to a greater ing of how pre-B cells integrate signals to cations for T lymphocytes and lympho- degree than the cells that received wild- continue proliferating or arrest in G1 cyte transformation. type CDK4. Furthermore, the inhibitory phase and initiate IgL gene assembly to effect of BTG2 co-expression with CDK4 promote the differentiation of mature B CONFLICT OF INTEREST was blunted in cells expressing the cells. The authors have identified a post- The authors declare no conflict of interest. CDK44R-K mutant. These findings translational mechanism that physically uncover a unique mechanism where hinders CDK4 from associating with ACKNOWLEDGEMENTS BTG2/PRMT1 promotes arginine Cyclin D3, thereby leading to G1 arrest, This work was supported by the Immune methylation of CDK4 to disrupt its RAG expression and IgL gene assembly. System Development and Regulation T32- association with Cyclin D3, thereby inhi- These findings may have broader impli- AI055428 (to GSW) and the National biting pre-B-cell proliferation and pro- cations for B-cell development, function Institutes of Health R01 grants AI112621 and moting B-cell differentiation. and transformation. Although Cyclin AI112621 (to CHB). Finally, Dolezal et al.1 investigated D3/CDK4 complexes drive proliferation whether BTG2 has a role in B-lineage of pro-B and pre-B cells, a pool of Cyclin cancers given that several B malignancies D3 functions independent of CDK4 to arise from dysregulated Cyclin D3/CDK4 downregulate transcription of IgH and 1 Dolezal E, Infantino S, Drepper F, Börsig T, κ 11 Singh A, Wossning T et al. The BTG2- activity. Indeed, BTG2 is a tumor- Ig variable (V) gene segments. PRMT1 module limits pre-B cell expansion suppressor gene and is frequently Accordingly, BTG2/PRMT1-mediated by regulating the CDK4-Cyclin-D3 complex. mutated in pre-B acute lymphoblastic destruction of CDK4 may suppress IgL Nat Immunol 2017; 18:911–920. 10 2 Winkler GS. The mammalian anti-prolifera- leukemia (ALL). A common lesion translocations by arresting pre-B cells in tive BTG/Tob protein family. 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Ubiquityla- these observations are also true in vivo, proliferating GC B cells to increase the tion of RAG-2 by Skp2-SCF links destruction the authors sorted Igκ− pre-B cells, efficiency of IgH class switch recombina- of the V(D)J recombinase to the cell cycle. Mol Cell 2005; 18:699–709. transformed them with BCR-ABL1, tion. If so, GC-like diffuse large B-cell 9 Cooper AB, Sawai CM, Sicinska E, Powers SE, transduced the cells with BTG2 or lymphomas may harbor mutations in Sicinski P, Clark MR et al. A unique func- BTG2ΔBoxC and then injected them BTG2 or PRMT1. Finally, the potential tion for cyclin D3 in early B cell develop- fi ment. Nat Immunol 2006; 7: 489–497. into immunode cient recipient mice. rolesofBTG2andPRMT1indeveloping 10 Mao B, Zhang Z, Wang G. BTG2: a rising star The BTG2ΔBoxC cells expanded and αβ T cells should be investigated as of tumor suppressors (review). 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