Vprbp (DCAF1) Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation

The Journal of Immunology

VprBP (DCAF1) Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation

N. Max Schabla, Greg A. Perry, Victoria L. Palmer, and Patrick C. Swanson

The assembly of Ig genes in developing B lymphocytes by V(D)J recombination is initiated by the RAG1–RAG2 endonuclease complex. We previously identified an interaction between RAG1 and viral protein R binding protein (VprBP) (also known as DNA damage binding protein 1 cullin 4–associated factor 1 [DCAF1]), a substrate receptor for the cullin 4–really interesting new gene (RING) E3 ubiquitin ligase (CRL4). We report in this article that in mice, B cell–intrinsic loss of VprBP increases RAG1 protein levels and disrupts expression of the endoribonuclease Dicer, which is essential for microRNA maturation. Rag1/2 transcription is known to be derepressed by loss of microRNA-mediated suppression of phosphatase and tensin homolog, raising the possibility that the elevated level of RAG1 observed in VprBP-deficient B cells is caused indirectly by the loss of Dicer. However, we show that VprBP restrains RAG1 expression posttranscriptionally and independently of Dicer. Specifically, loss of VprBP stabilizes RAG1 protein, which we show is normally degraded via a mechanism requiring both 20S proteasome and cullin–RING E3 ubiquitin ligase activity. Furthermore, we show that RAG1 stabilization through small molecule inhibition of cullin–RING E3 ubiquitin ligase activation promotes V(D)J recombination in a murine pre–B cell line. Thus, in addition to identifying a role for VprBP in maintaining Dicer levels in B cells, our findings reveal the basis for RAG1 turnover and provide evidence that the CRL4VprBP(DCAF1) complex functions to maintain physiological levels of V(D)J recombination. The Journal of Immunology, 2018, 201: 930–939.

he generation of a diverse lymphocyte Ag receptor rep- and lymphocyte development remains incomplete. The discovery ertoire is fundamental to adaptive immunity. Developing that the RAG1 NTR contains a functional really interesting new T B cells assemble functional Ig genes from arrays of var- gene (RING) domain (10), which is characteristic of E3 ubiquitin iable, diverse, and joining segments via an orchestrated process of ligases that catalyze the transfer of ubiquitin to substrate proteins DNA cleavage and joining called V(D)J recombination (1). The (11), raised the possibility that RAG1 mediates ubiquitylation of process is initiated by RAG1 and RAG2, which together compose itself and/or other target proteins to facilitate V(D)J recombina- the site-specific endonuclease that introduces DNA double strand tion. Indeed, evidence supporting both possibilities has been breaks at the recombination signal sequences flanking Ig gene published; RAG1 is reported to undergo autoubiquitylation (12), segments (2, 3). The N-terminal region (NTR) of RAG1, spanning which may stimulate its V(D)J recombination activity (12–14), aa 1–383, is dispensable for RAG catalytic activity in vitro (4, 5). and has also been implicated in mediating ubiquitylation of other However, deletion of the NTR impairs the efficiency and fidelity proteins, including the nuclear transport protein karyopherin-a of V(D)J recombination and reduces peripheral B and T cell (15) and histone H3 (16, 17). However, the relationship between numbers in vivo (6–9). substrate ubiquitylation and V(D)J recombination in vivo remains A mechanistic understanding for how the RAG1 NTR con- unclear. tributes to maintaining normal levels of V(D)J recombination A second, non–mutually exclusive functional role for the NTR is as a protein–protein interaction domain used to recruit accessory factors that enhance or regulate V(D)J recombination Department of Medical Microbiology and Immunology, Creighton University, (18–21). The functional significance of these interactions is not Omaha, NE 68178 fully understood. We previously identified an association between Received for publication January 11, 2018. Accepted for publication June 3, 2018. the RAG1 NTR and viral protein R binding protein (VprBP, also This work was supported by National Institutes of Health (NIH) Grant R01GM102487 (to P.C.S.) and by revenue from Nebraska’s excise tax on cigarettes known as DNA damage binding protein 1 cullin 4–associated awarded to Creighton University through the Nebraska Department of Health & factor 1 [DCAF1], but henceforth referred to as VprBP), a substrate Human Services. NIH funding to support research laboratory construction (Grant receptor for the cullin 4–RING E3 ubiquitin ligase (CRL4) (21). We C06 RR17417-01), the Creighton University Animal Resource Facility (Grant G20RR024001), the acquisition of the GE Healthcare Typhoon 9410 Variable Mode further showed that when mb1-Cre transgene expression (22) is used Imager (Grant S10RR027352), and the Bio-Rad ZE5 Cell Analyzer (Grant to conditionally inactivate Vprbp in murine B cells (VprBPdel/del 3R01GM102487-03S1) is gratefully acknowledged. mice), B cell development is blocked at the pro– to pre–B cell The contents of this publication represent the views of the authors and do not nec- transition, and distal V –DJ and Vk–Jk gene rearrangement is essarily represent the official views of the State of Nebraska Department of Health & H H Human Services or the National Institutes of Health. impaired (21). Address correspondence and reprint requests to Dr. Patrick C. Swanson, Department More recently, we reported that the developmental block in del/del of Medical Microbiology and Immunology, Creighton University, 2500 California VprBP mice could be partially bypassed by enforced ex- Plaza, Omaha, NE 68178. E-mail address: [email protected] pression of Bcl2 (VprBPdel/del Bcl2+ mice), which also restored Abbreviations used in this article: BAFF, B cell activating factor; BM, bone marrow; distal V gene rearrangement at both the Igh and Igk loci (23). CHX, cycloheximide; CRL, cullin–RING E3 ubiquitin ligase; CRL4, cullin 4–RING del/del E3 ubiquitin ligase; DGCR8, DiGeorge critical region 8; Dicerfl/fl,B6.Cg-Dicer1tm1Bdh/J; Interestingly, most B cells reaching the periphery in VprBP + + miRNA, microRNA; NAE, NEDD8-activating E1 enzyme; NTR, N-terminal region; Bcl2 mice are Igl , reflecting an ∼10-fold loss in the absolute PTEN, phosphatase and tensin homolog; qRT-PCR, quantitative RT-PCR; RING, really number of splenic Igk+ B cells. This outcome is correlated with interesting new gene; RS, recombining sequence; VprBP, viral protein R binding protein. increased levels of secondary V(D)J rearrangements in the Igk Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 locus, including k-deletion and skewing of Vk rearrangements to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800054 The Journal of Immunology 931

Jk5 as well as elevated rearrangement of the Igl locus (23). These cages in an American Association for the Accreditation of Laboratory secondary V(D)J rearrangements are often associated with re- Animal Care–certified facility at Creighton University. For all experiments, ceptor editing, which can be initiated in response to ligation of the control and knockout animals were either cohoused littermates or were de- rived from parallel breedings. All experimental procedures were reviewed BCR by self-antigen at the immature B cell stage (24), raising the and approved by the Creighton University Institutional Animal Care and Use possibility that loss of VprBP in VprBPdel/del Bcl2+ mice leads to Committee. excessive V(D)J recombination and receptor editing. The skewing of the B cell repertoire toward Igl+ B cells ob- BM culture served in VprBPdel/delBcl2+ mice is also reminiscent of what is Mice were sacrificed by cervical dislocation at 6–8 wk of age, and BM cells observed in Bcl2-transgenic mice with a B lineage deficiency in were flushed from the femora and tibiae. Cells were prepared as described the DiGeorge critical region 8 (DGCR8) protein (25), a compo- (29). Briefly, adherent cells were removed by incubating collected BM cells at 37˚C for 15 min. Nonadherent cells were collected by centrifugation, nent of the microprocessor complex essential for producing mature subjected to RBC lysis in ACK Lysing Buffer (Thermo Fisher Scientific), microRNAs (miRNAs) (26). This outcome was traced to derepression washed, and seeded at a density of 7.5 3 105 cells/ml in 13 IMDM sup- of Rag expression and excessive receptor editing caused indirectly by plemented with 10% FBS (Life Technologies), 13 penicillin/streptomycin 25 the loss of miRNA-mediated silencing of phosphatase and ten- (Corning), 2 mM L-glutamine (Sigma-Aldrich), 5.5 3 10 M 2-ME m sin homolog (PTEN), a lipid phosphatase that antagonizes PI3K (Sigma-Aldrich), and 10 ng/ l of recombinant murine IL-7 (R&D Systems). Cells were cultured at 37˚C with 5% CO2 for two consecutive periods of 4 d, signaling (25). with cells being reseeded at their initial density after the first culture period. Consistent with the possibility that excessive V(D)J recombi- For experiments including IL-7 withdrawal, cells were washed twice in nation is a causal factor that explains the phenotype observed in 10 ml of culture medium and reseeded at 7.5 3 105 cells/ml in fresh m VprBPdel/delBcl2+ mice, we show in this study that RAG1 protein medium containing 20 ng/ l recombinant murine B cell activating factor (BAFF; R&D Systems) and cultured 2 d prior to analysis. levels are elevated in bone marrow (BM) B cells cultured from del/del + VprBP Bcl2 mice. In the same cells, we show a severe re- Flow cytometry and cell sorting duction in the protein level of the endoribonuclease Dicer, another Single-cell suspensions prepared from BM or spleen of 6- to 8-wk-old mice factor which is essential for miRNA biogenesis (26). This raised or cultured BM cells were analyzed by multicolor immunophenotyping. To the possibility that the skewed Igk/Igl ratio and excessive receptor detect surface Ags, cells were Fc-blocked using anti-CD16/32 (2.4G2; editing observed in VprBPdel/delBcl2+ mice is a secondary effect Becton Dickenson) and stained with the following fluorochrome-conjugated caused by Dicer loss in developing B cells. However, the increase Abs: eBioscience anti–TCRb-PE (H57-597), anti–CD19-AlexaFluor700 in RAG1 protein under these conditions was not correlated with (eBio1D3), anti–IgM-PE-Cy5 (Il/41), anti–CD93-PE-Cy7 (AA4.1), Becton- Dickinson anti–B220-PE-CF594 (RA3-6B2), anti–CD43-allophycocyanin elevated Rag1 transcript levels. Furthermore, although we ob- (S7), anti–Igk-PerCP-Cy5.5 (187.1), anti–CD21/CD35-allophycocyanin served increased levels of Rag1 transcript and RAG1 protein in (7G6), biotinylated anti-CD23 (B3B4) detected with BV510-streptavidin, splenic B cells from Bcl2-transgenic mice with a B lineage– anti–Igl-FITC (R26-46), and BioLegend anti–IgD-allophycocyanin-Cy7 specific deficiency in Dicer (Dicerdel/delBcl2+ mice), which is (11-26c.2a). For detecting intracellular PTEN, cells were fixed and permeabilized after surface staining using the Cytofix/Cytoperm kit (Becton consistent with results reported by Coffre et al. (25), splenic Dickinson) according to manufacturer instructions. Permeabilized cells were del/del + B cells from VprBP Bcl2 mice showed no increase in Rag1 stained with anti–PTEN-AlexaFluor488 (IC847G; Novus Biologicals) or transcript, yet RAG1 protein remained elevated. Moreover, RAG1 isotype control Ab (MOPC-21; eBioscience). All samples were stained with protein levels were not elevated in cultured BM B cells from violet-fluorescent LIVE/DEAD dye (Life Technologies) for discrimination Dicerdel/delBcl2+ mice. Instead, we found that loss of VprBP of nonviable cells. All phenotypic analyses were performed using a ZE5 Analyzer (Bio-Rad Laboratories). For these analyses, B cells were defined as expression extended the half-life of the RAG1 protein and that live, B220+CD19+TCRb2 cells within a lymphocyte gate. For sorting of turnover of RAG1 was proteasome-dependent and required Nedd8- splenic B cells, splenocytes were stained with Becton-Dickenson Abs anti– dependent activation of cullin–RING E3 ubiquitin ligases (CRLs). B220-PE (RA3-6B2), anti–CD19-allophycocyanin-Cy7 (1D3), and anti– + + 2 These results suggest a model in which VprBP regulates RAG1 CD25-allophycocyanin-R700 (PC61) and B220 CD19 CD25 Bcellswere sorted using a FACSAria (Becton Dickinson). Sorting of BM was performed turnover by directing its proteasomal degradation via the activated after staining cells with Becton Dickenson Abs anti–B220-PE (RA3-6B2), anti– CRL4 complex. IgM-PE-Cy5 (Il/41), and anti–CD43-biotin (S7) detected using BUV737- We conclude that although both VprBP and Dicer negatively conjugated streptavidin. Pre–B cells were sorted as B220+CD432IgM2.All data generated in phenotyping and sorting experiments were analyzed using the regulate RAG1 expression to restrain excessive RAG activity, they fl/fl + do so through distinct mechanisms; VprBP controls RAG1 expression FlowJo software package (Tree Star). For cell cycle analysis, VprBP Bcl2 and VprBPdel/delBcl2+ BM cultures were subjected to B cell enrichment by posttranslationally via proteasome-dependent degradation, whereas magnetic sorting (Miltenyi Biotec) to remove residual non–B cells. Purified Dicer suppresses Rag transcription. We speculate that these mech- B cells were resuspended in Vindelov’s reagent (100 mM Tris, 0.9% NaCl, anisms operate at different stages of B cell ontogeny, with VprBP 0.01 mg/ml RNase A, 0.075 mg/ml propidium iodide, 0.1% Nonidet P-40) being crucial for suppressing RAG1 in pre–B and immature B cells and analyzed on the ZE5. Cell cycle modeling was performed using the ModFit program (Verity Software House). and Dicer functioning to control RAG expression in peripheral B cells. Our findings reveal previously unidentified roles for VprBP Western blot analysis in maintaining Dicer protein levels during B cell development and in Cultured or sorted cells (2–4 3 106) were incubated for 10 min on ice in regulating V(D)J recombination by mediating RAG1 turnover. RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with 1 mM Materials and Methods sodium orthovanadate and 2% v/v protease inhibitor mixture (P8340; Sigma- Mice Aldrich). Proteins were resolved by SDS-PAGE prior to transfer onto PVDF membrane and detection. The following primary Abs were used: anti-Dicer The generation of VprBPfl/flBcl2+ and VprBPdel/delBcl2+ mice has been (A301-936A; Bethyl Laboratories), anti-VprBP (11612-I-AP; Proteintech described (23, 27). Mice bearing conditional Dicer alleles (28) (B6.Cg- group), anti-RAG1 [rabbit mAb developed by the Schatz group, see (18)], Dicer1tm1Bdh/J; Dicerfl/fl) were purchased from The Jackson Laboratory. To anti-RAG2 (MABE1104; EMD Millipore), and anti–b-actin (A5316; Sigma- generate Dicerdel/delBcl2+ mice and littermate controls, Dicerfl/fl mice were Aldrich). HRP-conjugated goat anti-rabbit or anti-mouse secondary Abs initially crossed with VprBPfl/flBcl2+ and VprBPdel/del mice to breed on the (Cell Signaling Technology) were used to detect primary Ab, and blots were Bcl2 or mb1-Cre transgene onto the Dicerfl background and then succes- developed using Pierce ECL 2 substrate (Thermo Fisher Scientific) and sively back-crossed to remove the conditional Vprbp alleles. The resulting imaged using a Typhoon 9410 Variable Mode Imager (GE Healthcare). All Dicerfl/flBcl2+ and Dicerdel/del mice were crossed to generate mice for Western blot signal quantification was performed using the ImageJ image analysis. All mice were housed in individually ventilated microisolator processing tool (https://imagej.NIH.gov). 932 VprBP (DCAF1) REGULATES RAG1 HALF-LIFE

Quantitative RT-PCR analysis cultured under both conditions (Fig. 1B); IL-7 withdrawal led to an Total RNA was isolated from cultured BM cells or sorted splenic B cells increase in the percentage of cells in the G1 phase of the cell cycle for using the GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich). both genotypes. Western blotting of cell lysates prepared from both cDNA was prepared using TaqMan Reverse Transcription Reagents (Life the 8d+IL-7 and the 2d+BAFF BM cultures revealed the presence Technologies). Quantitative PCRs were set up using 23 SYBR Green of VprBP in samples from VprBPfl/flBcl2+ mice but not VprBPdel/del Master Mix (Thermo Fisher Scientific) and carried out on a CFX96 Real- Bcl2+ mice (Fig. 1C). Interestingly, RAG1 protein was elevated Time system (Bio-Rad Laboratories) with the following thermal cycling ∼ del/del + conditions: 95˚C for 1 min, followed by 35 cycles of 95˚C for 30 s, 55˚C for 15 s, 6-fold in 8d+IL-7 BM cultures from VprBP Bcl2 mice rel- fl/fl + and 72˚C for 15 s. Data were analyzed using software accompany- ative to VprBP Bcl2 mice (Fig. 1C, 1D), but levels of Rag1 ing the instrument. The following primer pairs were used for quantitative RT- transcript were not significantly different in these samples (Fig. 1E). PCR (qRT-PCR) assays (30, 31) (MP203654; OriGene Technologies): Withdrawal of IL-7 stimulated a ∼10-fold increase in Rag1 tran- Rag1 forward 59-ATGGCTGCCTCCTTGCCGTCTACC-39; Rag1 reverse, fl/fl + 9 9 9 script in VprBP Bcl2 cells, which was accompanied by a ∼3-fold 5 -CTGAGGAATCCTTCTCCTTCTGTG-3 ; Rag2 forward, 5 -TTAATT- del/ CCTGGCTTGGCCG-39; Rag2 reverse, 59-TTCCTGCTTGTGGATGTGAAAT- increase in RAG1 protein levels (Fig 1C–E). By contrast, VprBP 39; Dicer forward, 59-AGCAGTGCTGAGAAGAGGAAGG-39;Dicer delBcl2+ cells failed to robustly induce Rag1 transcription, showing reverse, 59-CCGCTTTTCTCCACAGTGATGC-39;Hprtforward,59- significantly lower expression than VprBPfl/flBcl2+ cells after IL-7 9 9 CTGGTGAAAAGGACCTCTCG-3 ; Hprt reverse, 5 -TGAAGTACTCAT- withdrawal (Fig. 1E). Notably, despite poor Rag1 transcriptional TATAGTCAAGGGCA-39. induction, RAG1 protein levels in these samples were ∼3-fold PCR Southern blot analysis of V(D)J rearrangements higher than in comparable samples from VprBPfl/flBcl2+ mice Genomic DNAwas prepared from sorted splenic B cells, and specific V(D)J (Fig. 1C, 1D). In these samples, RAG2 protein levels were rearrangements were amplified from 10,000, 2,500, or 625 cell equivalents similar between VprBP-proficient and VprBP-deficient cells (Fig. 1C), in PCRs with primer sets and thermal cycling conditions as previously whereas the levels of Rag2 transcript followed a similar pattern as described (23). For PCR amplification of Vk–recombining sequence (RS) observed for Rag1 (Fig. 1E). rearrangements, the RS primer (23) was paired with a degenerate Vk primer [59-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-39 (32)], Recent work by the Koralov group (25) suggests that Rag expression and thermal cycling conditions identical to those for intron RS–RS were is constrained at the transcriptional level indirectly through miRNA- used (23). PCR products were transferred to nitrocellulose membranes, mediated suppression of PTEN. They further reported that loss of 32 hybridized with P-labeled oligonucleotide probes, and detected by miRNAs in B cells skews the ratio of Igk+/Igl+ cells toward Igl+ autoradiography. B cells, a feature that resembles what we previously observed in RAG1 protein half-life experiments VprBPdel/delBcl2+ mice (23). Two reports indicating a role for VprBP in directing ubiquitin-dependent degradation of Dicer in response to BM cells from VprBPfl/flBcl2+ or VprBPdel/delBcl2+ mice were expanded 8 d in the presence of IL-7, followed by 48 h of culture in medium lacking certain stimuli (36, 37) suggested a potential mechanistic link between IL-7, and supplemented with BAFF as described above. At 24 h following Rag expression and VprBP through its regulation of Dicer-mediated IL-7 withdrawal, MLN4924 (3 mM; Selleckchem), bortezomib (5 mM; miRNA biogenesis. This prompted us to investigate whether Dicer Selleckchem), or DMSO (vehicle control) was added, and cells were expression was altered in VprBP-deficient B cell progenitors. cultured for an additional 24 h. Cycloheximide (CHX, 20 mg/ml; Sigma- Strikingly, we found that Dicer is undetectable by Western blot Aldrich) was added for the final 2 h of the culture period, and cells were del/del + harvested at 30 min intervals. For experiments using the A70 cell line (33), in VprBP Bcl2 BM cells cultured under both the 8d+IL-7 and cells were seeded at a density of 1 3 106 cells/ml in RPMI medium sup- 2d+BAFF conditions (Fig. 1C). The effect of VprBP deficiency on plemented with 10% FBS (Life Technologies), 13 penicillin/streptomycin Dicer protein levels is independent of transcription, as there is no 25 (Corning), 2 mM L-glutamine (Sigma-Aldrich), and 5.5 3 10 M 2-ME fl/fl m significant difference in Dicer mRNA levels between VprBP (Sigma-Aldrich). Cells were cultured with STI-571 (5 M; Tocris Biosci- + del/del + ence) for 24 h and then cultured for an additional 24 h in the presence Bcl2 and VprBP Bcl2 BM cultured under either condition of MLN4924 (3 mM) or DMSO (vehicle control). For the final 2 h, CHX (Fig. 1F). Taken together, these data suggest that VprBP suppresses (20 mg/ml) was added, and cells were harvested at 30 min intervals. RAG1 expression posttranscriptionally and is necessary for efficient Statistics transcriptional upregulation of Rag1 upon attenuation of IL-7 receptor signaling. Furthermore, VprBP is necessary for maintaining Data are presented as mean values 6 SEM. All data were analyzed by cellular Dicer levels in a manner that is independent of Dicer ANOVA with Bonferroni Correction using the GraphPad Prism platform. The p values #0.05 were considered significant. transcription. VprBP and Dicer restrain excessive RAG activity through Results distinct mechanisms VprBP controls expression of RAG1 and Dicer in The previous data suggest that the increase in RAG1 protein ob- developing B cells served in VprBPdel/delBcl2+ B cells is not a secondary effect of Our previous observation that k-deletion was elevated in sorted Dicer loss that results in Rag transcriptional upregulation. To Igk+ B cells from VprBPdel/delBcl2+ mice (23) raised the possi- test this more directly, we generated Dicerfl/flBcl2+ and Dicerdel/ bility that the skewed Igk+/Igl+ ratio on this genetic background is delBcl2+ mice as reported previously (25, 38) and compared caused by excessive RAG activity. To explore this possibility, we them to VprBPfl/flBcl2+ and VprBPdel/delBcl2+ mice (Fig. 2). used a stromal cell–independent BM culture system in which B220+CD19+ B cells account for ∼60 and ∼40% of live TCRb2 pro– and pre–B cells are expanded in the presence of IL-7 for 8 d splenocytes in VprBPdel/delBcl2+ and Dicerdel/delBcl2+ mice, (8d+IL-7) and then cultured in the presence of BAFF without IL-7 for respectively (Fig. 2A, top panels). The percentage of B cells expressing an additional 2 d (2d+BAFF) (29). IL-7 withdrawal has been shown surface Ig was higher in VprBPdel/delBcl2+ mice compared with to stimulate transit from the large to small pre–B cell stage and up- Dicerdel/delBcl2+ mice (Fig. 2A, middle panels). Nevertheless, the ab- regulate Rag expression, whereas BAFF enhances cell survival in the solute number of splenic B cells and the absolute numbers of Igk+ absence of IL-7 (29, 34, 35). Compared to 0 d cultures, the 8d+IL-7 and Igl+ B cells were not significantly different between these two BM cultures from VprBPfl/flBcl2+ and VprBPdel/delBcl2+ mice genetic backgrounds (Fig. 2B, left and right panels, respectively). showed substantial enrichment of B220+CD19+ cells, which persisted Consistent with the phenotype of Bcl2-transgenic DGCR8-deficient in the 2d+BAFF cultures (Fig. 1A). Cells from VprBPfl/flBcl2+ and B cells reported previously (25), splenic B cells from Dicerdel/del VprBPdel/delBcl2+ mice showed very similar cell cycle profiles when Bcl2+ mice express low levels of surface IgM, but this feature is not The Journal of Immunology 933

FIGURE 1. VprBP controls expression of Dicer and RAG1 in developing B cells. (A) BM cells from VprBPfl/flBcl2+ and VprBPdel/delBcl2+ mice were cultured for 0 or 8 d in the presence of IL-7 (0 or 8d+IL7) with or without subsequent IL-7 withdrawal and BAFF supplementation for 2 d (2d+BAFF). Cells were analyzed by flow cytometry to detect surface expression of B220 and CD19. The percentages of gated B220+CD19+ cells are representative of multiple experiments. (B) B cells enriched by magnetic sorting were stained with Vindelov’s reagent for determination of cell cycle status. Percentage of cells in each cell cycle phase is shown. (C) Lysates prepared from cultured BM cells were subjected to SDS-PAGE and Western blotting to detect the indicated proteins. (D) Western blot signal for RAG1 was quantified and normalized to the b-actin signal, with signal from the VprBPfl/flBcl2+ 8d+IL-7 sample arbitrarily set to 1.0. Data are representative of two independent experiments. (E and F) RNA isolated from cultured BM cells was subjected to qRT-PCR to analyze transcript levels of Rag1 and Rag2 (E)orDicer (F), using Hprt levels for normalization. Error bars represent the mean 6 SEM from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant. recapitulated in splenic B cells from VprBPdel/delBcl2+ mice expression of PTEN, which suppresses PI3K signaling and (Fig. 2C). thereby allows elevated transcription and activity of Ragsinthe In VprBPdel/delBcl2+ and Dicerdel/delBcl2+ mice, the ratio of periphery. Although we observed increased levels of intracellular Igk+/Igl+ cells was observed to be ∼1:3 and ∼1:1, respectively, PTEN in splenic B cells from both VprBPdel/delBcl2+ mice and whereas it was ∼3:1 in both VprBPfl/flBcl2+ and Dicerfl/flBcl2+ Dicerdel/delBcl2+ mice using flow cytometry (Fig. 2F, 2G), Rag1 mice (Fig. 2A bottom panels and Fig. 2D). As might be expected, and Rag2 transcript levels were only found to be elevated in levels of intron RS–to-RS and Vk-RS rearrangements leading to sorted B cells from Dicerdel/delBcl2+ mice and not VprBPdel/del k-deletion (39) were correspondingly increased in sorted B220+CD19+ Bcl2+ mice, consistent with the block in Rag transcription ob- CD252 splenic B cells from both VprBPdel/delBcl2+ and Dicerdel/del served in cultured BM cells from the latter mice (Fig. 2H). Bcl2+ mice compared with their counterparts lacking mb1-Cre but Despite low levels of Rag1 transcript in VprBPdel/delBcl2+ were slightly higher in the sorted B cells from VprBPdel/delBcl2+ mice splenic B cells, RAG1 protein was clearly detected by Western than those from Dicerdel/delBcl2+ mice (Fig. 2E). Excessive L chain blotting in these cells, whereas it was not evident in VprBPfl/fl gene rearrangement in these mice was also evidenced by skewing of Bcl2+ cells (Fig. 2I). Consistent with increased Rag1 transcript Jk gene segment usage to Jk5 as well as increased Vl1–Jl1 in Dicerdel/delBcl2+ splenic B cells, RAG1 was also detectable in rearrangement (Fig. 2E). these cells, albeit at a lower level than in VprBPdel/delBcl2+ cells The Koralov group (25) attributed the excessive Ig L chain (Fig. 2I). Taken together, these data suggest that although both gene editing phenotype of DGCR8-deficient B cells to increased VprBP and Dicer function to constrain V(D)J recombination 934 VprBP (DCAF1) REGULATES RAG1 HALF-LIFE

FIGURE 2. VprBP and Dicer restrain Rag1 expression and receptor editing through distinct mechanisms. (A) Total splenocytes prepared from mice of the indicated genotypes were analyzed by flow cytometry for surface expression of CD19, B220, IgM, IgD, Igk,andIgl.(B) Total numbers of splenocytes, splenic lymphocytes, and splenic B cells (left panel) and total numbers of splenic Igk+ and Igl+ B cells (right panel) were determined for each genotype (n = 6–13; listed at right) and displayed in bar graph format. Error bars represent the mean 6 SEM. (C) Representative plot comparing surface IgM (sIgM) staining for splenic B cells from mice of the indicated genotypes. (D) The proportion of Igk+ and Igl+ cells among total L chain–bearing splenic B cells was determined for the mice analyzed in (B) and is presented in bar graph format. (E)SplenicB220+CD19+CD252 B cells sorted from mice of the indicated genotypes were subjected to PCR and Southern blotting to amplify and detect specific V(D)J rearrangements listed to the right of each panel from 10,000, 2,500, or 625 cell equivalents of genomic DNA. Amplification of the nonrearranging CD14 locus serves as a loading control. (F) Splenocytes from mice of the indicated genotypes were analyzed by intracellular flow cytometry to detect PTEN expression. Staining with an isotype control Ab is shown (light blue peak). (G) Quantitation of the mean fluorescence intensity (MFI) was determined for each genotype (right panel; n = 6–13). ***p , 0.001 by one-way ANOVAwith Bonferroni post hoc correction. (H) Total RNA from splenic B cells sorted as in (A) was subjected to qRT-PCR to detect Rag1 and Rag2 transcript. The level of Rag1 and Rag2 in VprBPfl/flBcl2+ thymocytes was arbitrarily set to 1.0 for comparison. (I) Lysates prepared from sorted splenic B220+CD19+CD252 B cells were probed by Western blotting as in Fig. 1C. *p , 0.05, **p , 0.01, ***p , 0.001 by one-way [or two-way for (G)] ANOVA. ns, not significant. IRS, intron recombining sequence. activity by controlling RAG protein levels, they do so via distinct Dicerdel/delBcl2+ mice were enriched to a similar degree (Fig. 3A). mechanisms. Importantly, whereas the level of RAG1 protein was elevated in cultured VprBPdel/delBcl2+ BM cells as compared with their Loss of Dicer alone is insufficient to elevate RAG1 expression VprBPfl/flBcl2+ counterparts, it was diminished in Dicerdel/del in cultured BM cells Bcl2+ cells (Fig. 3B), suggesting that elevation of RAG1 protein is Given the upregulation of Rag1 transcript observed in splenic not secondary to the loss of Dicer on the VprBPdel/delBcl2+ B cells from Dicerdel/delBcl2+ mice but not VprBPdel/delBcl2+ background. Furthermore, we found that levels of Rag1 and Rag2 mice, we next wished to determine whether this phenomenon transcript were slightly diminished in both the VprBP- and Dicer- would also be observed in the BM cell culture system. In the 8d + deficient BM cultures (Fig. 3C). Intracellular PTEN levels were IL-7 cultures, B220+CD19+ cells from VprBPdel/delBcl2+ and not elevated in either knockout strain, suggesting that miRNAs are The Journal of Immunology 935

FIGURE 3. Loss of Dicer alone is insufficient to elevate RAG1 expression in cultured BM cells. (A) BM cells from mice of the indicated genotypes were cultured in the presence of IL-7 and analyzed as in Fig. 1A. Data are representative of three independent experiments. (B) Lysates prepared from 8d+IL-7 BM cultures were probed by Western blotting as in Fig. 1B. Data are representative of two independent experiments. (C) Rag1 and Rag2 transcript levels were compared between cultured BM cells from the indicated mice as in Fig. 1E. (D and E) Intracellular PTEN was detected by flow cytometry, and the mean fluorescence intensity (MFI) for PTEN staining was quantified as in Fig. 2F and 2G. (F) V(D)J rearrangements in sorted BM pre–B cells (B220+ CD432IgM2) were analyzed by PCR and southern blotting as in Fig. 2E. Error bars represent the mean 6 SEM for three independent experiments. IRS, intron recombining sequence. not critical for suppressing Rag expression at early B cell devel- rearrangement in Dicerdel/delBcl2+ pre–B cells as compared with opmental stages (Fig. 3D, 3E). Consistent with these observations, Dicerfl/flBcl2+ counterparts (Fig. 3F), which may be partly at- marks of receptor editing, including k-deletion, Igl rearrange- tributed to lower RAG1 protein levels observed in Dicerdel/del ment, and skewing of Vk–Jk joins to Jk5, were elevated in sorted Bcl2+ BM cultures (Fig. 3B) (see Discussion). Taken together, VprBPdel/delBcl2+ BM pre–B cells as compared with VprBPfl/fl these data support a model in which, during early B cell develop- Bcl2+ pre–B cells, whereas levels of these rearrangements be- ment, VprBP functions to posttranscriptionally regulate RAG1 tween Dicer-proficient and -deficient pre–B cells were quite protein levels independently of its role in maintaining cellular Dicer similar (Fig. 3F). We also noted a marked overall decrease in Igk levels, whereas in the periphery, Dicer is necessary for proper 936 VprBP (DCAF1) REGULATES RAG1 HALF-LIFE suppression of Rag transcription, but loss of VprBP overrides the treated with CHX for the final 2 h of the culture period to block effects of an absence of Dicer. all protein translation. Lysates prepared from cells harvested at 30 min intervals during CHX treatment were probed for RAG1 VprBP Is required for normal turnover of RAG1 protein protein by immunoblotting. Under these conditions, we observed We next sought to determine the mechanism by which VprBP that RAG1 was degraded with an estimated half-life of ∼30 min mediates RAG1 posttranscriptional suppression. A well-established in VprBPfl/flBcl2+ cells. (Fig. 4A). In striking contrast, RAG1 feature of cellular RAG1 is its short half-life of ∼10–30 min (40–42). protein levels in VprBPdel/delBcl2+ cells remained consistently We speculated that loss of VprBP might extend RAG1 half-life, elevated through the entire 2 h CHX treatment period, indicating leading to its accumulation. To test this possibility, 2d+BAFF BM that VprBP is required for normal turnover of the RAG1 protein cultures from VprBPfl/flBcl2+ and VprBPdel/delBcl2+ mice were (Fig. 4A).

FIGURE 4. Loss of VprBP or inhibition of the proteasome or NAE activity extends RAG1 half-life and promotes increased V(D)J recombination. (A)(Top panel) BM cells from VprBPfl/flBcl2+ or VprBPdel/delBcl2+ mice were cultured for 8 d in the presence of IL-7 and then subjected to IL-7 withdrawal and BAFF supplementation for 2 d. For the final 2 h of the culture period, CHX was added (20 mg/ml), and cells were harvested at 30 min intervals. Cell lysates were analyzed by Western blotting as in Fig. 1C. Western blot signal for RAG1 was quantified and normalized to the b-actin signal as in Fig. 1D, and relative RAG1 signal at each time point was normalized to the sample at time 0. (B and C) BM cells from VprBPfl/flBcl2+ mice were cultured as in (A) and treated during the final 24 h (after IL-7 withdrawal) with vehicle (DMSO), MLN4924 [3 mM(B)], or bortezomib [5 mM(C)], with CHX added during the final 2 h of culture. RAG1 and RAG2 protein levels were analyzed by Western blotting and quantified as in (A). (D) A70 cells were incubated in the presence of STI-571 for 48 h and treated during the final 24 h with vehicle (DMSO) or MLN4924, with CHX added during the final 2 h of culture as in (B)and(C). RAG1 and RAG2 were detected and quantified by Western blotting as in (B)and(C). (A–D) Error bars represent mean 6 SEM for two independent experiments. (E) Diagram of the pMX-INV reporter substrate. (F) A70 cells were treated with vehicle and inhibitor combinations as in (D) and analyzed by flow cytometry to detect GFP expression. Representative histograms from each treatment are shown along with quantification of the percentage of GFP+ cells from three independent experiments. Error bars represent mean 6 SEM from three independent experiments. ***p , 0.001 by one-way ANOVA with Bonferroni post hoc correction. The Journal of Immunology 937

This observation, along with VprBP’s established role as a The short half-life of RAG1 may be a strategy by which substrate receptor for the CRL4 E3 ubiquitin ligase (reviewed in developing lymphocytes restrain RAG activity to limit off-target Ref. 43), raised the possibility that VprBP directs the degradation cleavage to protect genomic stability. Our observation that loss of RAG1 via the CRL4VprBP complex. To test this possibility, we of VprBP is associated with an increase in Ig L chain gene re- repeated the experiment with VprBPfl/flBcl2+ cells as shown in combination events and loss of Igk+ B cells also suggests that this Fig. 4A, except that cells were treated during the last 24 h of the mechanism contributes to the proper developmental timing of Igk IL-7 withdrawal period with MLN4924, an inhibitor of the Nedd8- and Igl rearrangement. This is an attractive model for two reasons. activating E1 enzyme (NAE) (44). The rationale for using this First, evidence suggests RAG2 is expressed in molar excess to inhibitor is that NAE is required for activating CRL complexes by RAG1 (∼15-fold); therefore, one would expect the latter to be initiating conjugation of the ubiquitin-like protein Nedd8 to the rate-limiting for V(D)J recombination (50). Indeed, our finding cullin scaffold (45–47). We found that MLN4924 treatment of that concomitant treatment of A70 cells with STI-571 and MLN4924 VprBPfl/flBcl2+ cells treated under these conditions showed no increases RAG1 levels, but not RAG2 levels, and promotes greater V apparent RAG1 degradation during the period of concomitant (D)J rearrangement of the GFP reporter substrate in these cells is CHX treatment, essentially mirroring the effect of loss of VprBP consistent with this possibility. The finding that Rag1 heterozy- (Fig. 4B). As a control, VprBPfl/flBcl2+ cells treated with vehicle gosity reduces receptor editing in a mouse model of enforced only (DMSO) showed similar turnover kinetics as observed with autoreactivity (51) also provides additional experimental support for VprBPfl/flBcl2+ cells in Fig. 4A. Furthermore, RAG2 levels remained this possibility. largely unchanged during the CHX treatment period and were not Second, the short half-life of RAG1 may provide a means to affected by concomitant exposure to MLN4924 (Fig. 4B). In keeping restrain V(D)J recombination during a phase of the cell cycle when with CRL4VprBP-dependent turnover of RAG1, we found that RAG2 levels are fixed. Specifically, RAG2 is known to undergo treatment of VprBPfl/flBcl2+ cells with the proteasome inhibitor phosphorylation-dependent ubiquitylation and degradation at the bortezomib (48) extended the half-life of RAG1 to ∼130 min G1-to-S phase transition but is stabilized during G1, the phase of compared with ∼30 min in the vehicle-treated control (Fig. 4C). the cell cycle to which V(D)J recombination is restricted (52–54). To further extend mechanistic studies of RAG1 turnover, we With CRLs being implicated in the turnover of RAG1, physio- investigated the effects of MLN4924 treatment in an Abelson logical mechanisms may exist to suppress CRL activity during the murine leukemia virus–transformed pre–B cell line called A70 G1 phase to enable transient upregulation of RAG1 levels. Fur- (33). Treatment of Abelson murine leukemia virus–transformed thermore, with VprBP also being implicated in controlling Rag pre–B cells with STI-571, a small molecule inhibitor of the vAbl transcriptional upregulation (Fig. 1), it is possible that VprBP levels kinase, induces G1 cell cycle arrest and stimulates Rag tran- are modulated to titrate Rag transcript levels during G1 as well. scription (49). Recapitulating the effects of MLN4924 treatment Coordination of these mechanisms may enable fine control of V(D)J observed with primary B cells from VprBPfl/flBcl2+ mice, we recombination activity within the G1 phase of the cell cycle and found that NAE inhibition in STI-571–treated A70 cells stabilizes would help explain how sequential V(D)J rearrangements, such as RAG1 in the presence of CHX, whereas RAG1 is turned over those occurring during receptor editing of the Ig L chain loci, are normally in A70 cells treated with STI-571 and DMSO as the facilitated and regulated within G1 without requiring a cell cycle vehicle control (Fig. 4D). Furthermore, the expression and half- transition to suppress V(D)J recombination through the targeted life of RAG2 remained unaffected by MLN4924 treatment in degradation of RAG2. STI-571–treated A70 cells (Fig. 4D). The skewing of the B cell repertoire in VprBPdel/delBcl2+ mice The A70 cells used in these experiments harbor a chromosomally toward Igl+ B cells, the association of this phenotype with in- integrated V(D)J recombination reporter substrate consisting of an creased k-deletion and skewed Vk rearrangement to Jk5, and the antisense cDNA encoding GFP flanked by a pair of recombination subsequent finding that RAG1 protein levels are elevated in cul- signal sequences, called pMX-INV (33). Upregulation of RAGs tured B cells from these animals are reminiscent of findings from upon STI-571 treatment enables inversion and expression of the the Koralov group (25) showing that loss of DGCR8 leads to antisense GFP cDNA (Fig. 4E). Treatment of A70 cells si- transcriptional upregulation of Rag expression and increased re- multaneously with STI-571 and MLN4924 resulted in an ∼25% ceptor editing due to loss of miRNA-mediated suppression of increase in expression of the GFP reporter substrate compared PTEN. We were therefore interested in determining whether the with cells treated with STI-571 and DMSO vehicle control, phenotypes observed in VprBPdel/delBcl2+ mice could be attrib- indicating that the increased half-life of RAG1 promotes in- uted to a secondary effect of the loss of Dicer. Although loss creased V(D)J recombination (Fig. 4F). Taken together, these of VprBP expression does lead to loss of Dicer (the implications data suggest that VprBP mediates RAG1 turnover through a of which are discussed further below), our direct comparison of mechanism that is proteasome-dependent and requires CRL VprBPdel/delBcl2+ mice and Dicerdel/delBcl2+ mice rules out this activity. possibility by showing that loss of VprBP in VprBPdel/delBcl2+ mice does not lead to transcriptional upregulation of Rag ex- Discussion pression and that loss of Dicer in Dicerdel/delBcl2+ mice does not Although long recognized, the mechanistic basis for the short half- increase RAG1 protein levels in cultured BM cells from these life of cellular RAG1 has remained obscure. Our findings have animals. Our observation that RAG1 is present in VprBPdel/del provided significant new insight that helps explain this phenom- Bcl2+ splenic B cells despite Rag1 transcript levels being com- enon, establishing that VprBP, which we previously identified as parable to VprBPfl/flBcl2+ cells (Fig. 2I) most likely reflects per- associating with full-length RAG1 (21), mediates RAG1 degra- sistence of RAG1 from earlier developmental stages resulting dation through a mechanism that is proteasome-dependent and from a complete loss of RAG1 turnover. Taken together, these data requires NAE activity. Because NAE-dependent neddylation of argue that VprBP and Dicer regulate RAG1 expression through cullins is required for their activation, the simplest conclusion is distinct mechanisms; Dicer controls RAG1 expression indirectly that VprBP promotes RAG1 degradation via the CRL4VprBP at the transcriptional level via miRNA-mediated suppression of complex. What remains to be clarified is the precise mechanism PTEN, and VprBP controls RAG1 expression at the posttran- by which RAG1 is directed to the proteasome for degradation. scriptional level by mediating its proteasome-dependent turnover. 938 VprBP (DCAF1) REGULATES RAG1 HALF-LIFE

Furthermore, these distinct mechanisms may operate at different Disclosures stages of B cell development, with regulation of PTEN expression The authors have no financial conflicts of interest. by miRNAs being critical in transitional and mature B cells, but not in the BM. This is consistent with the observation that cultured BM cells from Dicerdel/delBcl2+ mice do not exhibit evidence of References 1. Schatz, D. G., and P. C. Swanson. 2011. V(D)J recombination: mechanisms of Rag transcriptional upregulation, as well as previous studies initiation. Annu. Rev. Genet. 45: 167–202. showing that deletion of PTEN does not rescue early B cell de- 2. Schatz, D. G., M. A. Oettinger, and D. Baltimore. 1989. The V(D)J recombi- velopment in mice lacking miRNAs that target Pten mRNA (55). nation activating gene, RAG-1. Cell 59: 1035–1048. 3. Oettinger, M. A., D. G. Schatz, C. Gorka, and D. Baltimore. 1990. RAG-1 and This notion is further supported by our observation that although RAG-2, adjacent genes that synergistically activate V(D)J recombination. del/del + marks of receptor editing are elevated in both VprBP Bcl2 Science 248: 1517–1523. and Dicerdel/delBcl2+ splenic B cells, this is only the case for BM 4. Silver, D. P., E. Spanopoulou, R. C. Mulligan, and D. Baltimore. 1993. Dis- pensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J pre–B cells of the former knockout strain (Figs. 2E, 3F). Our recombination. Proc. Natl. Acad. Sci. USA 90: 6100–6104. experiments also revealed an overall reduction in Vk–Jk rear- 5. Kirch, S. A., P. Sudarsanam, and M. A. Oettinger. 1996. Regions of RAG1 rangements in Dicerdel/delBcl2+ pre–B cells in the absence of a protein critical for V(D)J recombination. Eur. J. Immunol. 26: 886–891. l l 6. Roman, C. A., S. R. Cherry, and D. Baltimore. 1997. Complementation of V(D)J large effect on V –J rearrangement (Fig. 3F). A study from the recombination deficiency in RAG-1(-/-) B cells reveals a requirement for novel Schlissel group (56) reported that expression of miR-290-5p and elements in the N-terminus of RAG-1. Immunity 7: 13–24. miR-292-5p is induced at the pro– to pre–B cell transition and that 7. Sekiguchi, J. A., S. Whitlow, and F. W. Alt. 2001. Increased accumulation of hybrid V(D)J joins in cells expressing truncated versus full-length RAGs. Mol. these miRNAs promote germline transcription of the Igk locus, Cell 8: 1383–1390. which is strongly correlated with initiation of Igk rearrangement 8. Dudley, D. D., J. Sekiguchi, C. Zhu, M. J. Sadofsky, S. Whitlow, J. DeVido, (32). Thus, it is possible that loss of miR-290-5p and miR-292-5p R. J. Monroe, C. H. Bassing, and F. W. Alt. 2003. Impaired V(D)J recombination k k del/del + and lymphocyte development in core RAG1-expressing mice. J. Exp. Med. 198: results in impaired V –J rearrangement in Dicer Bcl2 pre– 1439–1450. B cells and may even contribute to the skewing in the Igk/Igl ratio 9. Talukder, S. R., D. D. Dudley, F. W. Alt, Y. Takahama, and Y. Akamatsu. 2004. on the miRNA-deficient genetic background (Fig. 2) (25). Further Increased frequency of aberrant V(D)J recombination products in core RAG- expressing mice. Nucleic Acids Res. 32: 4539–4549. work will be required to investigate these possibilities. 10. Yurchenko, V., Z. Xue, and M. Sadofsky. 2003. The RAG1 N-terminal domain is Interestingly, despite the absence of Dicer and the elevation of an E3 ubiquitin ligase. Genes Dev. 17: 581–585. PTEN in splenic B cells from VprBPdel/delBcl2+ mice, we did not 11. Deshaies, R. J., and C. A. Joazeiro. 2009. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78: 399–434. observe any increase in Rag1 or Rag2 transcript. This finding is 12. Jones, J. M., and M. Gellert. 2003. Autoubiquitylation of the V(D)J recombinase also consistent with the absence of Rag transcriptional upregula- protein RAG1. Proc. Natl. Acad. Sci. USA 100: 15446–15451. tion upon IL-7 withdrawal in BM cultures from VprBPdel/delBcl2+ 13. Simkus, C., A. Bhattacharyya, M. Zhou, T. D. Veenstra, and J. M. Jones. 2009. Correlation between recombinase activating gene 1 ubiquitin ligase activity and mice (Fig. 1E). These data together suggest that VprBP has a V(D)J recombination. Immunology 128: 206–217. secondary role in regulating Rag transcription downstream of 14. Singh, S. K., and M. Gellert. 2015. Role of RAG1 autoubiquitination in V(D)J PTEN/PI3K. The mechanism underlying this observation remains recombination. Proc. Natl. Acad. Sci. USA 112: 8579–8583. 15. Simkus, C., M. Makiya, and J. M. Jones. 2009. Karyopherin alpha 1 is a putative to be determined. substrate of the RAG1 ubiquitin ligase. Mol. Immunol. 46: 1319–1325. Finally, the finding that loss of VprBP leads to a profound, 16. Grazini, U., F. Zanardi, E. Citterio, S. Casola, C. R. Goding, and F. McBlane. transcription-independent loss of Dicer protein in B cell progen- 2010. The RING domain of RAG1 ubiquitylates histone H3: a novel activity in chromatin-mediated regulation of V(D)J joining. Mol. Cell 37: 282–293. itors contrasts strikingly with reports that VprBP targets Dicer for 17. Jones, J. M., A. Bhattacharyya, C. Simkus, B. Vallieres, T. D. Veenstra, and degradation via the CRL4VprBP complex in response to certain M. Zhou. 2011. The RAG1 V(D)J recombinase/ubiquitin ligase promotes stimuli (36, 37). If this model were true in B cells, one might have ubiquitylation of acetylated, phosphorylated histone 3.3. Immunol. Lett. 136: 156–162. expected that Dicer protein levels would increase upon loss of 18. Coster, G., A. Gold, D. Chen, D. G. Schatz, and M. Goldberg. 2012. A dual VprBP. The discrepancy between our findings and these previous interaction between the DNA damage response protein MDC1 and the RAG1 reports may be attributed to the involvement of specific stimuli to subunit of the V(D)J recombinase. J. Biol. Chem. 287: 36488–36498. 19. Maitra, R., and M. J. Sadofsky. 2009. A WW-like module in the RAG1 N- induce Dicer degradation that are not recapitulated in the cultured terminal domain contributes to previously unidentified protein-protein interac- BM cells used in this work. Specifically, in the study by Klockow tions. Nucleic Acids Res. 37: 3301–3309. et al. (36), Dicer degradation was shown to be caused by hijacking 20. Raval, P., A. N. Kriatchko, S. Kumar, and P. C. Swanson. 2008. Evidence for VprBP Ku70/Ku80 association with full-length RAG1. Nucleic Acids Res. 36: 2060– of the CRL4 complex by the HIV-1 accessory factor Vpr. 2072. Whether all Vpr-directed substrates are also physiological sub- 21. Kassmeier, M. D., K. Mondal, V. L. Palmer, P. Raval, S. Kumar, G. A. Perry, strates of CRL4VprBP remains unclear, and thus it is possible that D. K. Anderson, P. Ciborowski, S. Jackson, Y. Xiong, and P. C. Swanson. 2012. VprBP binds full-length RAG1 and is required for B-cell development and V(D) VprBP only mediates Dicer degradation in the context of HIV J recombination fidelity. EMBO J. 31: 945–958. infection (57, 58). In the study by Ren et al. (37), Dicer degra- 22. Hobeika, E., S. Thiemann, B. Storch, H. Jumaa, P. J. Nielsen, R. Pelanda, and dation was elicited by stimulation of HCT116 colon cancer cells M. Reth. 2006. Testing gene function early in the B cell lineage in mb1-cre mice. Proc. Natl. Acad. Sci. USA 103: 13789–13794. with TLR ligands and IL-6. Thus, it is possible that VprBP 23. Palmer, V. L., R. Aziz-Seible, M. D. Kassmeier, M. Rothermund, G. A. Perry, functions to maintain Dicer levels via a mechanism that is re- and P. C. Swanson. 2015. VprBP is required for efficient editing and selection of stricted to B cells or lymphocytes. Further studies will be needed Igk+ B cells, but is dispensable for Igl+ and marginal zone B cell maturation and selection. J. Immunol. 195: 1524–1537. to investigate these possibilities. In any case, given the pleotropic 24. Nemazee, D. 2017. Mechanisms of central tolerance for B cells. Nat. Rev. roles attributed to VprBP (reviewed in Ref. 43) and the findings Immunol. 17: 281–294. reported in this article and elsewhere (36, 37) that the Dicer levels 25. Coffre, M., D. Benhamou, D. Rieß, L. Blumenberg, V. Snetkova, M. J. Hines, T. Chakraborty, S. Bajwa, K. Jensen, M. M. W. Chong, et al. 2016. miRNAs are may be regulated by VprBP, some caution is warranted in inter- essential for the regulation of the PI3K/AKT/FOXO pathway and receptor preting results of experiments in which VprBP levels are sup- editing during B cell maturation. Cell Rep. 17: 2271–2285. pressed using knockdown or knockout approaches due to potential 26. Winter, J., S. Jung, S. Keller, R. I. Gregory, and S. Diederichs. 2009. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat. Cell Biol. secondary effects caused by changes in cellular Dicer levels. 11: 228–234. 27. McCall, C. M., P. L. Miliani de Marval, P. D. Chastain, II, S. C. Jackson, Acknowledgments Y. J. He, Y. Kotake, J. G. Cook, and Y. Xiong. 2008. Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the We thank Dr. Sleckman for providing A70 cells and Dr. Schatz and DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embry- Elizabeth Corbett for providing anti-RAG1 Ab. onic development. Mol. Cell. Biol. 28: 5621–5633. The Journal of Immunology 939

28. Harfe, B. D., M. T. McManus, J. H. Mansfield, E. Hornstein, and C. J. Tabin. 43. Nakagawa, T., K. Mondal, and P. C. Swanson. 2013. VprBP (DCAF1): a pro- 2005. The RNaseIII enzyme Dicer is required for morphogenesis but not pat- miscuous substrate recognition subunit that incorporates into both RING-family terning of the vertebrate limb. Proc. Natl. Acad. Sci. USA 102: 10898–10903. CRL4 and HECT-family EDD/UBR5 E3 ubiquitin ligases. BMC Mol. Biol. 14: 22. 29. Holl, T. M., B. F. Haynes, and G. Kelsoe. 2010. Stromal cell independent B cell 44. Soucy, T. A., P. G. Smith, M. A. Milhollen, A. J. Berger, J. M. Gavin, development in vitro: generation and recovery of autoreactive clones. J. Immu- S. Adhikari, J. E. Brownell, K. E. Burke, D. P. Cardin, S. Critchley, et al. 2009. nol. Methods 354: 53–67. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. 30. Hassaballa, A. E., V. L. Palmer, D. K. Anderson, M. D. Kassmeier, Nature 458: 732–736. V. K. Nganga, K. W. Parks, D. L. Volkmer, G. A. Perry, and P. C. Swanson. 2011. 45. Duda, D. M., L. A. Borg, D. C. Scott, H. W. Hunt, M. Hammel, and Accumulation of B1-like B cells in transgenic mice over-expressing catalytically B. A. Schulman. 2008. Structural insights into NEDD8 activation of cullin- inactive RAG1 in the periphery. Immunology 134: 469–486. RING ligases: conformational control of conjugation. Cell 134: 995–1006. 31. Amin, R. H., and M. S. Schlissel. 2008. Foxo1 directly regulates the transcription 46. Jones, J., K. Wu, Y. Yang, C. Guerrero, N. Nillegoda, Z. Q. Pan, and L. Huang. of recombination-activating genes during B cell development. Nat. Immunol. 9: 2008. A targeted proteomic analysis of the ubiquitin-like modifier nedd8 and 613–622. associated proteins. J. Proteome Res. 7: 1274–1287. 32. Schlissel, M. S., and D. Baltimore. 1989. Activation of immunoglobulin kappa 47. Pan, Z. Q., A. Kentsis, D. C. Dias, K. Yamoah, and K. Wu. 2004. Nedd8 on gene rearrangement correlates with induction of germline kappa gene tran- cullin: building an expressway to protein destruction. Oncogene 23: 1985–1997. scription. Cell 58: 1001–1007. 48. Adams, J., V. J. Palombella, E. A. Sausville, J. Johnson, A. Destree, 33. Bredemeyer, A. L., G. G. Sharma, C. Y. Huang, B. A. Helmink, L. M. Walker, D. D. Lazarus, J. Maas, C. S. Pien, S. Prakash, and P. J. Elliott. 1999. Proteasome K. C. Khor, B. Nuskey, K. E. Sullivan, T. K. Pandita, C. H. Bassing, and inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 59: B. P. Sleckman. 2006. ATM stabilizes DNA double-strand-break complexes 2615–2622. during V(D)J recombination. Nature 442: 466–470. 49. Muljo, S. A., and M. S. Schlissel. 2003. A small molecule Abl kinase inhibitor 34. Ochiai, K., M. Maienschein-Cline, M. Mandal, J. R. Triggs, E. Bertolino, induces differentiation of Abelson virus-transformed pre-B cell lines. Nat. R. Sciammas, A. R. Dinner, M. R. Clark, and H. Singh. 2012. A self-reinforcing Immunol. 4: 31–37. regulatory network triggered by limiting IL-7 activates pre-BCR signaling and 50. Zhang, Y. H., K. Shetty, M. D. Surleac, A. J. Petrescu, and D. G. Schatz. 2015. Nat. Immunol. differentiation. 13: 300–307. Mapping and quantitation of the interaction between the recombination acti- 35. Johnson, K., T. Hashimshony, C. M. Sawai, J. M. R. Pongubala, J. A. Skok, vating gene proteins RAG1 and RAG2. J. Biol. Chem. 290: 11802–11817. I. Aifantis, and H. Singh. 2008. Regulation of immunoglobulin light-chain re- 51. Verkoczy, L., D. Aı¨t-Azzouzene, P. Skog, A. Ma¨rtensson, J. Lang, B. Duong, and combination by the transcription factor IRF-4 and the attenuation of interleukin- D. Nemazee. 2005. A role for nuclear factor kappa B/rel transcription factors in 7 signaling. Immunity 28: 335–345. the regulation of the recombinase activator genes. Immunity 22: 519–531. 36. Klockow, C. L., H. J. Sharifi, X. Wen, M. Flagg, A. K. M. Furuya, 52. Li, Z., D. I. Dordai, J. Lee, and S. Desiderio. 1996. A conserved degradation M. Nekorchuk, and C. M. C. de Noronha. 2013. The HIV-1 protein Vpr targets signal regulates RAG-2 accumulation during cell division and links V(D)J re- the endoribonuclease Dicer for proteasomal degradation to boost macrophage Immunity infection. Virology 444: 191–202. combination to the cell cycle. 5: 575–589. 37. Ren, W., S. Shen, Z. Sun, P. Shu, X. Shen, C. Bu, F. Ai, X. Zhang, A. Tang, 53. Jiang, H., F. C. Chang, A. E. Ross, J. Lee, K. Nakayama, K. Nakayama, and L. Tian, et al. 2016. Jak-STAT3 pathway triggers DICER1 for proteasomal S. Desiderio. 2005. Ubiquitylation of RAG-2 by Skp2-SCF links destruction of degradation by ubiquitin ligase complex of CUL4A(DCAF1) to promote colon the V(D)J recombinase to the cell cycle. Mol. Cell 18: 699–709. cancer development. Cancer Lett. 375: 209–220. 54. Fisher, M. R., A. Rivera-Reyes, N. B. Bloch, D. G. Schatz, and C. H. Bassing. 38. Koralov, S. B., S. A. Muljo, G. R. Galler, A. Krek, T. Chakraborty, 2017. Immature lymphocytes inhibit Rag1 and Rag2 transcription and V(D)J C. Kanellopoulou, K. Jensen, B. S. Cobb, M. Merkenschlager, N. Rajewsky, and recombination in response to DNA double-strand breaks. J. Immunol. 198: K. Rajewsky. 2008. Dicer ablation affects antibody diversity and cell survival in 2943–2956. the B lymphocyte lineage. Cell 132: 860–874. 55. Lai, M., A. Gonzalez-Martin, A. B. Cooper, H. Oda, H. Y. Jin, J. Shepherd, 39. Shimizu, T., T. Iwasato, and H. Yamagishi. 1991. Deletions of immunoglobulin L. He, J. Zhu, D. Nemazee, and C. Xiao. 2016. Regulation of B-cell development C kappa region characterized by the circular excision products in mouse and tolerance by different members of the miR-17∼92 family microRNAs. Nat. splenocytes. J. Exp. Med. 173: 1065–1072. Commun. 7: 12207. 40. Grawunder, U., D. G. Schatz, T. M. J. Leu, A. Rolink, and F. Melchers. 1996. 56. Garcia, P. B., A. Cai, J. G. Bates, H. Nolla, and M. S. Schlissel. 2012. miR290- The half-life of RAG-1 protein in precursor B cells is increased in the absence of 5p/292-5p activate the immunoglobulin kappa locus in B cell development. PLoS RAG-2 expression. J. Exp. Med. 183: 1731–1737. One 7: e43805. 41. Ochodnicka-Mackovicova, K., M. Bahjat, C. Maas, A. van der Veen, 57. Hrecka, K., M. Gierszewska, S. Srivastava, L. Kozaczkiewicz, S. K. Swanson, T. A. Bloedjes, A. M. de Bruin, H. van Andel, C. E. Schrader, R. W. Hendriks, L. Florens, M. P. Washburn, and J. Skowronski. 2007. Lentiviral Vpr usurps E. Verhoeyen, et al. 2016. The DNA damage response regulates RAG1/2 ex- Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc. Natl. pression in Pre-B cells through ATM-FOXO1 signaling. J. Immunol. 197: 2918– Acad. Sci. USA 104: 11778–11783. 2929. 58. Wen, X., K. M. Duus, T. D. Friedrich, and C. M. de Noronha. 2007. The HIV1 42. Sadofsky, M. J., J. E. Hesse, J. F. McBlane, and M. Gellert. 1993. Expression and protein Vpr acts to promote G2 cell cycle arrest by engaging a DDB1 and V(D)J recombination activity of mutated RAG-1 proteins. Nucleic Acids Res. 21: Cullin4A-containing ubiquitin ligase complex using VprBP/DCAF1 as an 5644–5650. adaptor. J. Biol. Chem. 282: 27046–27057.