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

B lymphocytes are vital components of vertebrate adaptive immune systems. However, B-cell dysfunction can promote autoimmunity and immunodeficiency, and errors in development can cause B-lineage lymphoid malignancies. B-cell development requires RAG1/RAG2 (RAG) endonuclease-mediated assembly of immunoglobulin (Ig) genes. The assembly of Ig heavy chain (IgH) genes in G1-phase pro-B cells produces IgH proteins that pair with VpreB/λ5 to form pre-B-cell receptor (pre-BCR) complexes. In pro-B cells, pre-BCR signaling inhibits Rag1/Rag2 transcription, stabilizes Cyclin D3 to drive cellular proliferation and promotes differentiation of large pre-B cells. Interleukin (IL)-7 augments this developmental transition by signaling through the IL-7 receptor (IL-7R) to inhibit transcription of Rag1/Rag2 and activate transcription of Cyclin D3 (Ccnd3). Cyclin D3 binds to and activates cyclin-dependent kinase 4 (CDK4) to drive G1-phase cells into the cell cycle (Figure 1a). Although IL-7 antagonizes Ig gene assembly and promotes pre-B-cell proliferation, pre-BCR signaling in pre-B cells has opposing effects by stimulating Rag1/Rag2 transcription and repressing Ccnd3 transcription. As pre-B cells lose contact with the IL-7 niche, pre-BCR signaling triggers G1 arrest and the assembly of Ig light chain (IgL) genes (Igκ or Igλ) (Figure 1b). In this research highlight, we discuss recent work from Dolezal et al.¹ that uncovers an important role for B-cell translocation gene (BTG2) and protein arginine methyl transferase 1 (PRMT1) in posttranslational inactivation of Cyclin D3/CDK4 complexes. This mechanism suppresses pre-B-cell proliferation and activates IgL gene assembly and pre-B-cell differentiation.¹

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.

BTG2 is an antiproliferative BTG/transducer of ErbB2 family member that inhibits the G1/S transition of the cell cycle.² BTG2 binds and activates PRMT1, forming complexes that methylate protein substrates.³ Prior experiments suggested that BTG2 and PRMT1 each had a role in promoting B-cell development;⁴ however, the mechanisms by which these two proteins regulated B-cell differentiation were not explored. Dolezal et al.¹ conducted experiments in mice and in cultured primary pre-B cells to determine the roles of BTG2 and PRMT1 in developing B cells. They found that B-lineage-specific deletion of Prmt1 resulted in a defect in early B-cell development as reflected by lower numbers of cells at the pro-B-cell stage onwards. Using a pre-B-cell line (1676) and ex vivo cultured primary pre-B cells, knockdown of Prmt1 mRNA in non-cycling cells resulted in a reduction of Igκ⁺ B cells, indicating that PRMT1 regulates pre-B-cell differentiation. Curiously, overexpression of Prmt1 did not enhance pre-B-cell development, suggesting other cellular factors limit PRMT1 activity. Given that PRMT1 and BTG2 complex in vitro,³ the authors quantified the BTG2 expression in developing B cells. They found that non-cycling 1676 pre-B cells exhibited elevated levels of Btg2 transcript and protein relative to cycling cells, mirroring the expression pattern of Prmt1. BTG2 contains a BoxC region that is required for BTG2 to bind and activate PRMT1.⁵ Overexpression of wild-type BTG2, but not a BTG2 mutant with a BoxC deletion (BTG2ΔBoxC), enhanced Igκ expression and pre-B-cell differentiation in 1676 and ex vivo cultured pre-B cells. These effects were dependent on PRMT1 as PRMT1 knockdown, even in the presence of overexpressed BTG2, reduced Igκ expression. BTG2 knockdown in 1676 and ex vivo cultured pre-B cells also reduced the frequency of Igκ⁺ cells. PRMT1 functions to asymmetrically dimethylate arginine residues (ADMA).⁶ Consistent with the cellular phenotype data, analysis of ADMA revealed extensive methylation in cells transduced with wild-type BTG2 but not the BTG2ΔBoxC mutant. Collectively, these data indicate that BTG2 and PRMT1 function together to promote pre-B-cell development.

Ig gene assembly occurs in G1-phase cells and requires the expression of both RAG1 and RAG2 proteins.⁷ Thus the authors next tested the effect of BTG2 on RAG1 and RAG2 expression in developing pre-B cells. 1676 pre-B cells transduced with wild-type BTG2 exhibited an increase in RAG2 protein as compared with control and BTG2ΔBoxC mutant cells. As RAG2 is degraded in S phase,⁸ Dolezal et al.¹ reasoned that the increase in RAG2 protein in BTG2-transduced cells was due to G1 cell cycle arrest. Consistent with this notion, the authors found that 1676 pre-B cells expressing wild-type BTG2 had a higher fraction of cells arrested in G1 phase. Given that IL-7R signaling promotes Cyclin D3 expression to drive cells through G1 and into S phase,⁹ the authors asked whether BTG2 and PRMT1 reduce Cyclin D3 expression. Indeed, overexpression of wild-type BTG2, but not the BTG2ΔBoxC mutant, resulted in a drastic reduction in Cyclin D3 protein in a proteasome-dependent manner. Conversely, BTG2 or PRMT1 knockdown increased Cyclin D3 protein and drove cell cycle progression. These data suggest that BTG2 and PRMT1 cooperate to arrest pre-B cells in G1 by licensing Cyclin D3 degradation. As Cyclin D3 fuels cell cycle progression, the authors hypothesized that Cyclin D3 is an inhibitor of the pre-B-cell differentiation program. Indeed, 1676 pre-B cells transduced with Cyclin D3 exhibited a decrease in the frequency of Igκ⁺ B cells. Conversely, knockdown of Cyclin D3 increased the frequency of Igκ⁺ 1676 pre-B cells. Notably, combined overexpression of Cyclin D3 and BTG2 led to a defect in the development of Igκ⁺ B cells, indicating that Cyclin D3 is a potent inhibitor of pre-B-cell differentiation. Collectively, these data reveal a critical role for the BTG2–PRMT1 complex in promoting Cyclin D3 degradation to suppress cell cycle progression, stimulate RAG expression and thereby promote pre-B-cell development.

Dolezal et al.¹ next assessed whether Cyclin D3 or CDK4 were targets of PRMT1-mediated methylation. They found that CDK4, but not Cyclin D3, was specifically methylated by PRMT1 using a fluorescent in vitro methylation assay. Moreover, CDK4 incubated with active PRMT1 and S-adenosyl methionine failed to bind Cyclin D3, indicating that Cyclin D3/CDK4 complexes are unstable when CDK4 is methylated by PRMT1. Analysis of CDK4 methylation by mass spectrometry revealed seven arginine residues that are targets of PRMT1, and of these, four residues (R55, R73, R82 and R163) were positioned at the surface where Cyclin D3 binds. To test whether these residues are required for PRMT1 to disrupt the Cyclin D3/CDK4 complex, the authors mutated all four arginines to lysine (CDK4^4R-K) and subjected the mutant protein to the fluorescent methylation and co-immunoprecipitation assays. As expected, the CDK4^4R-K mutant was refractory to PRMT1-mediated methylation compared with wild-type CDK4, resulting in its stable association with Cyclin D3. Presumably, the increased stability of Cyclin D3/CDK4^4R-K complexes would promote cell cycle progression. To test this idea, the authors overexpressed the CDK4^4R-K mutant in 1676 pre-B cells and measured growth. Indeed, the cells transduced with the CDK4^4R-K mutant expanded to a greater degree than the cells that received wild-type CDK4. Furthermore, the inhibitory effect of BTG2 co-expression with CDK4 was blunted in cells expressing the CDK4^4R-K mutant. These findings uncover a unique mechanism where BTG2/PRMT1 promotes arginine methylation of CDK4 to disrupt its association with Cyclin D3, thereby inhibiting pre-B-cell proliferation and promoting B-cell differentiation.

Finally, Dolezal et al.¹ investigated whether BTG2 has a role in B-lineage cancers given that several B malignancies arise from dysregulated Cyclin D3/CDK4 activity. Indeed, BTG2 is a tumor-suppressor gene and is frequently mutated in pre-B acute lymphoblastic leukemia (ALL).¹⁰ A common lesion observed in pre-B-ALL is a translocation that juxtaposes the breakpoint cluster region with the Abelson tyrosine kinase 1 gene (BCR-ABL1). The authors sought to determine whether BTG2 was able to suppress malignant pre-B-cell growth in BCR-ABL1-transformed pre-B cells. 1676 pre-B cells transduced with BCR-ABL1 exhibited a profound decrease in Btg2 mRNA and BTG2 protein expression, and this reduction was dependent upon the activity of BCR-ABL1. Transduction of BTG2, but not the BTG2ΔBoxC mutant, in BCR-ABL1-transformed 1676 pre-B cells resulted in a dramatic decrease in cellular proliferation. Notably, this decrease was lost when PRMT1 was knocked down, indicating that BTG2 and PRMT1 act together to restrict BCR-ABL1-induced proliferation. To determine whether these observations are also true in vivo, the authors sorted Igκ⁻ pre-B cells, transformed them with BCR-ABL1, transduced the cells with BTG2 or BTG2ΔBoxC and then injected them into immunodeficient recipient mice. The BTG2ΔBoxC cells expanded and were readily detected in the bone marrow and spleen of recipient animals. In stark contrast, the BTG2 cells were not detected in the bone marrow or spleen, indicating that in vitro and in vivo BTG2 and PRMT1 restricts the proliferation of BCR-ABL1-transformed pre-B cells.

This study advances our understanding of how pre-B cells integrate signals to continue proliferating or arrest in G1 phase and initiate IgL gene assembly to promote the differentiation of mature B cells. The authors have identified a posttranslational mechanism that physically hinders CDK4 from associating with Cyclin D3, thereby leading to G1 arrest, RAG expression and IgL gene assembly. These findings may have broader implications for B-cell development, function and transformation. Although Cyclin D3/CDK4 complexes drive proliferation of pro-B and pre-B cells, a pool of Cyclin D3 functions independent of CDK4 to downregulate transcription of IgH and Igκ variable (V) gene segments.¹¹ Accordingly, BTG2/PRMT1-mediated destruction of CDK4 may suppress IgL translocations by arresting pre-B cells in G1 before transcriptional repression of Cyclin D3 activates Igκ gene assembly. In response to DNA double-strand breaks (DSBs), pre-B cells transcriptionally repress Cyclin D3 levels,¹² which might be important to prevent G1-phase cells with DSBs from entering S phase where DSBs are prone to form translocations. It should be investigated whether DSBs signal via BTG2/PRMT1 to inactivate Cyclin D3/CDK4 complexes and thereby more rapidly halt the G1-to-S transition in response to DSBs. During an immune response, germinal center (GC) B cells require Cyclin D3 to rapidly proliferate and effectively perform IgH class switch recombination.¹³ According to ImmGen databases, both BTG2 and PRMT1 are expressed in GC B cells. Thus it possible that BTG2/PRMT1 posttranslationally regulates Cyclin D3/CDK4 complexes in proliferating GC B cells to increase the efficiency of IgH class switch recombination. If so, GC-like diffuse large B-cell lymphomas may harbor mutations in BTG2 or PRMT1. Finally, the potential roles of BTG2 and PRMT1 in developing αβ T cells should be investigated as Cyclin D3 drives cellular proliferation during DN-to-DP thymocyte differentiation and DP thymocytes express BTG2 and PRMT1. The BTG2/PRMT1 axis highlights a unique posttranslational mechanism that regulates pre-B-cell proliferation and differentiation. This mechanism may have important implications for T lymphocytes and lymphocyte transformation.

Conflict of interest

The authors declare no conflict of interest.