CUL7 E3 Ubiquitin Ligase Mediates the Degradation of Activation

CUL7 E3 Ubiquitin Ligase Mediates the Degradation of Activation-Induced Cytidine Deaminase and Regulates the Ig Class Switch Recombination in B Lymphocytes This information is current as of September 24, 2021. Yuewen Luo, Yang Liu, Liyang Wu, Xiancai Ma, Qin Liu, Feng Huang, Xu Zhang, Yiwen Zhang, Junsong Zhang, Haihua Luo, Yanyan Yang, Gen Lu, Xiaoping Tang, Linghua Li, Yixin Zeng, Ting Pan and Hui Zhang

J Immunol published online 15 May 2019 Downloaded from http://www.jimmunol.org/content/early/2019/05/14/jimmun ol.1900125 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/05/14/jimmunol.190012 Material 5.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 15, 2019, doi:10.4049/jimmunol.1900125 The Journal of Immunology

CUL7 E3 Ubiquitin Ligase Mediates the Degradation of Activation-Induced Cytidine Deaminase and Regulates the Ig Class Switch Recombination in B Lymphocytes

Yuewen Luo,*,†,‡,1 Yang Liu,x,1 Liyang Wu,*,†,‡ Xiancai Ma,*,†,‡ Qin Liu,{ Feng Huang,*,†,‡,‖ Xu Zhang,*,†,‡ Yiwen Zhang,*,†,‡ Junsong Zhang,*,†,‡ Haihua Luo,*,†,‡ Yanyan Yang,* Gen Lu,‖ Xiaoping Tang,# Linghua Li,# Yixin Zeng,x Ting Pan,*,†,‡ and Hui Zhang*,†,‡

Activation-induced cytidine deaminase (AID) initiates class switch recombination and somatic hypermutation in Ig genes. The activity and protein levels of AID are tightly controlled by various mechanisms. In this study, we found that CUL7 E3 ubiquitin ligases Downloaded from specifically mediated AID ubiquitination. CUL7 overexpression or knockdown influenced the decay of AID, affecting AID protein levels and subsequently IgA class switching in CH12F3 cells, a mouse B lymphocyte cell line. Further analysis indicated that CUL7 mediated AID ubiquitination by forming a complex with FBXW11. In a CUL7fl/flCD19cre+ mouse model, we demonstrated that CUL7 knockout significantly enhanced AID protein levels in B cells in the germinal center and increased both the IgG1 and IgA class switching. Collectively, our results reveal a subtle regulation mechanism for tightly controlling AID protein levels. The manipulation of this pathway may be useful for regulating AID abundance and efficiency of Ig class switching and is therefore a http://www.jimmunol.org/ potential target for developing immunologic adjuvants for vaccines of various pathogens such as HIV-1 and influenza viruses. The Journal of Immunology, 2019, 203: 000–000.

o fight microbial invasion, vertebrates must produce a and activity of AID are tightly regulated in cells at multiple layers, variety of high-affinity Abs. Limited Ab diversification including the transcription, posttranscription, and posttranslational T initially occurs through V(D)J recombination. The func- levels (5–8). AID is a nucleocytoplasmic shuttling protein (9). In the tions and diversification of Abs are further increased by class cytoplasm, AID typically interacts with HSP90 (10), eEF1A (11), switch recombination (CSR) and somatic hypermutation when and HSP40-DnaJa1 (12) to prevent self-degradation. Once AID

B cells enter the germinal centers. CSR and somatic hypermutation enters the nucleus passively (13, 14) or actively (15), excess nu- by guest on September 24, 2021 depend on the activity of activation-induced cytidine deaminase clear AID is exported to the cytoplasm by CRM1 (13) or quickly (AID) (1, 2). AID deaminates cytosines in both the donor and ac- degraded in the nucleus (16). It has been reported that AID is degraded ceptor S regions in H chain constant regions of Ab genes and by the proteasome through the REG-g–mediated ubiquitin-ATP– subsequently initiates CSR. The off-target effects of AID may ac- independent proteasome pathway (17) or ubiquitin-ATP–dependent tivate oncogenes such as c-myc by promoting translocation with proteasome pathway (16). However, the underlying mechanism re- IgH (3, 4). To avoid such disastrous consequences, the expression mains unclear.

*Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen Univer- China (Grant 31500740), the Natural Science Foundation of Guangdong (Grant sity, Guangzhou 510080, China; †Key Laboratory of Tropical Disease Control of 2015A030310442), the Science and Technology Planning Project of Guangzhou Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, (Grant 201704020226), and the Pearl River S&T Nova Program of Guangzhou Guangzhou 510080, China; ‡Guangdong Engineering Research Center for Antimi- (Grant 201806010118) (to T.P.); the National Natural Science Foundation of China (Grant crobial Agent and Immunotechnology, Zhongshan School of Medicine, Sun Yat-sen 81701990) (to F.H.); and the Natural Science Foundation of Guangdong (Grant University, Guangzhou 510080, China; xDepartment of Experimental Research, 2016A030313826) (to H.L.). Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South { Y. Luo designed and performed experiments, analyzed data, and wrote the manu- China, Guangzhou 510060, China; Guanghua School of Stomatology, Hospital of script. Y. Liu performed experiments, constructed plasmid, assisted with maintained Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of ‖ cells, mice, and plasmid preparation. X.M. and L.W. provided materials and exper- Stomatology, Guangzhou 510060, Guangdong, China; Department of Respiration, imental assistance. J.Z. and F.H. provided constructive suggestions and experimental Afﬁliated Guangzhou Women and Children’s Hospital, Zhongshan School of assistances. Q.L. provided materials and edited the language of manuscript. T.P., Y.Z., Medicine, Sun Yat-Sen University, Guangzhou 510080, China; and #Department X.Z., G.L., H.L., Y.Y., Y.Z., L.L., and X.T. provided assistance and constructive of Infectious Diseases, Guangzhou Eighth People’s Hospital, Guangzhou Medical suggestions for experiments. H.Z. conceived the project, supervised the work, ana- University, Guangzhou 510060, China lyzed data, and wrote the manuscript. 1These authors contributed equally. Address correspondence and reprint requests to Dr. Ting Pan and Dr. Hui Zhang, ORCIDs: 0000-0002-4934-4221 (X.M.); 0000-0001-9702-6167 (G.L.). Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen Uni- versity, Room 1308, New Technology Building, No. 74, Zhongshan 2nd Road, Received for publication February 1, 2019. Accepted for publication April 18, 2019. Guangzhou 510080, China. E-mail addresses: [email protected] (T.P.) and This work was supported by the National Special Research Program of China for [email protected] (H.Z.) Important Infectious Diseases (Grants 2018ZX10302103 and 2017ZX10202102), the The online version of this article contains supplemental material. Important Key Program of Natural Science Foundation of China (Grant 81730060), the International Collaboration Program of Natural Science Foundation of China, the Abbreviations used in this article: AID, activation-induced cytidine deaminase; National Institutes of Health (Grant 81561128007), and the Joint-Innovation Pro- Co-IP, coimmunoprecipitation; CRL, CUL-RING ligase; CSR, class switch recom- gram in Healthcare for Special Scientiﬁc Research Projects of Guangzhou (Grant bination; CUL, Cullin; IP, immunoprecipitation; siRNA, small interfering RNA. 201803040002) (to H.Z.); the National Science and Technology Major Project (Grant 2018ZX10101004003001) and the National Natural Science Foundation Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 of China (Grant 81601759) (to J.Z.); the National Natural Science Foundation of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900125 2 CUL7 REGULATES CSR BY UBIQUITINATING AND DEGRADING AID

The ubiquitin-proteasome system is one of the most impor- Materials and Methods tant biological systems in cells. The small peptide ubiquitin is Animals composed of 76 aa and conjugates to a substrate protein through a Mice were bred and housed in a specific pathogen-free environment at cascade of enzymatic reactions by two E1-activating enzymes, at Sun Yat-sen University Laboratory Animal Center in accordance with least 38 E2-conjugating enzymes, and over 600 E3 ligase en- the guidelines and principles of the Institutional Animal Care and Use Committee of Sun Yat-sen University. The following mouse strains zymes (18, 19). The proteasome recognizes the ubiquitin-linked tm1(cre)cgn substrate and mediates its degradation. E3 ligase enzymes can be were used in this study: B6.129P2(C)-Cd19 /J (JAX006785) and B6.129(Cg)-Cul7tm2.1Jdec/J (JAX018057). Experiments were per- divided into three main categories: RING, HECT, or RBR E3 formed using age 10–12-wk, littermate, and gender-matched mice in ligases. RING-domain E3 ligases bring the substrate to interact strict accordance with good animal practice as defined by the National with the ubiquitin-linked E2 enzyme and subsequently mediate Center for the Replacement, Refinement and Reduction of Animals in substrate ubiquitination (20). Both HECT-domain and RBR- Research. domain E3 ligases recruit the substrate and directly participate Cells in ubiquitin transfer (20). Numerous multisubunit RING-domain HEK293T cells were obtained from American Type Culture Collection and E3 enzymes have been identified, including the Cullin (CUL)- maintained in DMEM supplemented with 10% FBS (Life Technologies) RING ligases (CRLs) (21). CRLs, which include CUL1, CUL2, plus 1% penicillin-streptomycin (Life Technologies) at 37˚C with 5% CO2. CUL3,CUL4A,CUL4B,CUL5,andCUL7inmammals,me- CH12F3 cells and mice primary spleen cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, diate the degradation of ∼20% of proteins in cells (21, 22). and 2-ME (55 mM; Life Technologies) at 37˚C with 5% CO2. CH12F3 cell Covalent attachment of NEDD8, a small ubiquitin-like protein, is a gift from Dr. T. Honjo (Kyoto University, Japan) through Dr. W. Hu Downloaded from to CUL proteins is required for CRL ubiquitin ligase activity (Kyoto University) and Dr. F. Meng (Shanghai Institue of Biochemistry (23, 24). The small molecule compound MLN4924 can inhibit and Cell Biology). HEK 293T cells and CH12F3 cells were confirmed mycoplasma free. the attachment of NEDD8 to CUL proteins and inhibit the function of CRLs (25). Western blotting and coimmunoprecipitation In this study, we found that CUL7 E3 ligase specifically Cells were lysed in ice-cold RIPA lysis buffer containing a protease in- ubiquitinates AID and promotes its degradation. Suppression of hibitor mixture (Sigma-Aldrich) for 30 min at 4˚C. The lysates were http://www.jimmunol.org/ CUL7 in vitro and in vivo increased the protein levels of AID and subjected to Western blot. For coimmunoprecipitation (Co-IP), the CSR. We further found that the CUL7-FBXW11 complex me- 293T cells were cultured in a 60-mm plate and transfected with the indicated plasmids. Forty-eight hours later, the cells were collected and diated the degradation of AID. These studies reveal a novel disrupted using NP-40 lysis buffer containing a protease inhibitor mixture regulatory mechanism of AID protein stability. (Sigma-Aldrich) and PMSF for 30 min at 4˚C. The supernatant of cell by guest on September 24, 2021

FIGURE 1. siRNA screening for CUL family identified CUL7 as a specific E3 ligase for AID. (A and B) MLN4924 treatment upregulated AID in 293T cells. Plasmids expressing AID-GFP or AID-NESm (F193A, L196A)–GFP were transfected to 293T cells. Twenty-four hours after transfection, the cells were treated with DMSO, MG132 (10 mM), or MLN4924 (20 mM) for an additional 12 h. DMSO treatment served as a negative control, whereas MG132 was a positive control. The fluorescence-field micrograph image was obtained and processed in the same way. Scale bar, 200 mm. Images were quantified by ImageJ software. Data are representative of three independent experiments. Graph shows relative fluorescence signal of GFP after different treatments. (C) siRNA screening for CUL family that affects AID levels. Individual siRNA for CULs and a plasmid expressing AID-NESm-GFP (F193A, L196A) were cotransfected into 293T cells. Forty-eight hours posttransfection, the cells were digested by Trypsin-EDTA and resuspended in PBS for FACS analysis. The median GFP signal was quantified by flow cytometry. Data are represented as the mean 6 SEM. **p , 0.01, ***p , 0.001. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. CUL7 knockdown or overexpression influenced protein levels and ubiquitination of AID in 293T cells. (A) CUL7 knockdown upregulated AID levels in 293T cells. 293T cells were cotransfected with siRNA for CUL7 and AID-FLAG plasmid. The AID-FLAG levels and CUL7 knockdown efficiency were determined by Western blotting using anti-FLAG Abs and anti-CUL7 Abs. GAPDH was used as a loading control. Graph shows relative AID-FLAG protein levels after different treatments. (B) CUL7 overexpression downregulated AID levels in 293T cells. 293T cells were cotransfected with plasmids expressing AID-FLAG or CUL7-HA. Forty-eight hours posttransfection, the indicated transfected cells were treated with MG132 (10 mM) for additional 12 h. The protein levels of AID were determined by Western blot. GAPDH was used as a loading control. Graph shows relative AID-FLAG protein levels after different treatments. (C) CUL7 knockdown downregulated AID ubiquitination in 293T cells. 293T cells cotransfected with siNC or siCUL7 and plasmids expressing Ub-HA and AID-FLAG were treated with MG132 for 12 h before harvest. AID ubiquitination was analyzed by Co-IP with anti-FLAG Abs followed by Western blotting with anti-HA Abs. GAPDH was used as a loading control. Graph shows relative ubiquitin levels of AID after different treatments. (D) CUL7 overexpression upregulated AID ubiquitination in 293T cells. AID-FLAG– and Ub-HA–expressing plasmids were cotransfected with CUL7-HA–expressing plasmid, CUL7H1464P-HA–expressing plasmid, or vector into 293T cells. AID ubiquitination in cells was analyzed by Co-IP with anti-FLAG Abs, followed by Western blotting with anti-HA Abs. GAPDH was used as a loading control. Graph shows relative ubiquitin levels of AID after different treatments. (E) CUL7 specifically interacted with AID. 293T cells were transfected with AID-FLAG. CUL7-AID interaction was examined by Co-IP with anti-FLAG beads followed by Western blotting with anti-CUL7 Abs and anti-RBX1 Abs. GFP-FLAG was the IP control. Graph shows normalized levels of AID-FLAG–interacted protein. All data are representative of at least three experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 4 CUL7 REGULATES CSR BY UBIQUITINATING AND DEGRADING AID lysates was separated by centrifugation at 12,000 rpm for 15 min at 4˚C. SDS-PAGE and subsequent Western blotting. The membranes were immu- Anti-HA agarose beads (Sigma-Aldrich) or anti-FLAG agarose beads noblotted with indicated Abs and IRDye secondary Abs (LI-COR) and visu- (Sigma-Aldrich) were mixed with the supernatant and incubated at 4˚C alized with the Odyssey infrared imaging system (LI-COR). The following for 4 h or overnight. The immunoprecipitated samples were analyzed by Abs were used in this paper: mouse monoclonal anti-HA (MBL), rabbit Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. CUL7 knockdown or overexpression influenced AID decay. (A) Schematic of AID decay detection by photoactivatable fluorescent protein (PAFP). The 293T cells were cotransfected with Dendra2 fusion protein plasmid and the indicated siRNAs. After 48 h, the transfected 293T cells were exposed to 405-nm light for 2 min to convert the green form into the red form of Dendra2. The red fluorescence signal was collected at 0, 2, 4, 6, or 8 h after the exposure. The relative fluorescence signal at each time point was normalized to the fluorescence intensity at 0 h. (B) CUL7 knockdown decreased AID-NESm-Dendra2 degradation rate. siREG-g was used as a positive control. (C) CUL7 knockdown decreased AID-Dendra2 degradation rate. (D and E) CUL7 overexpression facilitated the degradation of AID-NESm-Dendra2 and AID-Dendra2. (F and G) CUL7 knockdown or overexpression had no impact on Dendra2 degradation. The signal data at the fourth hour after photoactivation were statistically analyzed. All above data are representative of at least three experiments. The Journal of Immunology 5 polyclonal anti-FLAG (MBL), rabbit polyclonal anti-GAPDH (Proteintech), fluorescence. To determine the decay rate of AID-dendra2, the intensity of rabbit polyclonal anti-CUL7 (Abcam), mouse monoclonal anti-AID (CST), red fluorescence was measured over time by fluorescence microscope mouse monoclonal anti–b-actin (Proteintech), rabbit polyclonal anti- (Leica). The images were analyzed by ImageJ software. FBXW11(Proteintech). All Western blot data were analyzed by ImageJ software. The relative protein levels were calculated by comparing nor- Ubiquitination assay malized mean gray value between different treatment and control. The transfected cells were lysed with RIPA buffer containing N-Ethylmaleimide Transfection (Sigma-Aldrich), PMSF, and protease inhibitor mixture after 8 h of MG132 (10 mM) (Selleckchem) treatment. The supernatant of cell lysates was Plasmid or chemically synthesized small interfering RNAs (siRNAs) separated by centrifugation at 12,000 rpm for 15 min at 4˚C. Anti-FLAG (RiboBio, Guangzhou, China) were transfected into 293T cells using agarose beads (Sigma-Aldrich) were mixed with the supernatant and in- Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. cubated at 4˚C for 4 h. Beads were then washed four times with 300 mM For CH12F3 cells, electrotransfection (Neon Transfection System; Thermo cold NaCl STN buffer and eluted with a gel loading buffer. The samples Fisher Scientific) was used according to the manufacturer’s protocol. were analyzed by SDS-PAGE, followed by Western blotting. Measurement of protein degradation with a photoactivatable Class switch assay fluorescent tag CD43-naive splenic B lymphocytes were purified by magnetic separation Green-to-red photoconvertible fluorescent protein Dendra2 was fused to (MACS; Miltenyi Biotec). Cells were maintained at 1–3 3 106 cells/ml C-terminal end of AID. 293T cells expressing AID-dendra2 were first in a standard conditioned medium and were treated with 5 ng/ml IL-4 exposed to 405-nm surface light (designed and produced by OPT Machine (PeproTech) and 25 mg/ml LPS (Sigma-Aldrich) so that CSR would be Vision Tech Co., LTD) for 2 min to convert green fluorescence to red induced for IgG1 generation, with 25 mg/ml LPS for induction of CSR to Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. CUL7 knockdown increased IgA class switch of CH12F3 B cells. (A) CUL7 knockdown increased IgA class switch of CH12F3 B cells. siNC or siCUL7 were electrotransfected into CH12F3 cells. After 12 h recovery, the transfected cells were stimulated with anti-CD40, IL-4, and TGF-b for 48 h. The frequency of IgA-positive cells was determined by ﬂow cytometry. Graph shows the percentage of IgA-positive cells. The data are representative of three experiments. (B) CUL7 knockdown increased AID levels in stimulated CH12F3 B cells. The protein levels of AID in CH12F3 B cells were determined by Western blot with anti-AID Abs. GAPDH was used as a loading control. Graph shows relative AID protein levels after different treatments. The data are representative of at least three experiments. (C–F) AICDA mRNA expression (C) and IgH gene germline transcription (E and F) were not inﬂuenced by CUL7 knockdown. The relative AICDA mRNA expression and germline transcription were determined by real-time quantitative PCR (n = 3). b-Actin was used as an internal reference. Data are reported as the mean 6 SEM. CUL7 knockdown increased circle transcripts in CH12F3 B cells (D). The relative expression of Ia-Cm circle transcripts were determined by real-time quantitative PCR (n = 3). HPRT was used as an internal reference. Data are reported as the mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. 6 CUL7 REGULATES CSR BY UBIQUITINATING AND DEGRADING AID Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. CUL7-FBXW11 complex-mediated AID degradation. (A) CUL7-promoted AID degradation was not inhibited by FBXW8 knockdown. siRNA for FBXW8 and plasmids expressing CUL7-HA or AID-FLAG were cotransfected into 293T cells. The protein levels of AID were determined by Western blot. GAPDH was used as loading control. Graph shows relative AID-FLAG protein levels after different treatments. (B) FBXW11 knockdown inhibited AID degradation that is mediated by CUL7 overexpression. The indicated siRNA for WD40 F-box E3 ligases family and AID-NESm-GFP (F193A, L196A) plasmid were cotransfected to 293T cells. Forty-eight hours posttransfection, the median GFP signal was acquired and quantified as described previously (Fig. 1C). (C) CUL7-mediated AID degradation was dependent on FBXW11. siRNA for FBXW11 and plasmids expressing CUL7-HA or AID-FLAG were cotransfected to 293T cells. The protein levels of AID were confirmed by Western blot. GAPDH was used as a loading control. Graph shows relative AID-FLAG protein levels after different treatments. (D) FBXW11 knockdown without CUL7 overexpression inhibited AID degradation. The siRNA for FBXW11 and plasmids expressing AID-FLAG were cotransfected to 293T cells. The AID-FLAG levels and FBXW11 knockdown efficiency were determined by Western blotting using anti-FLAG Abs and anti-FBXW11 Abs. GAPDH was used as a loading control. Graph shows relative AID-FLAG protein levels after different treatments. (E) FBXW11 overexpression promoted AID degradation in CUL7+/+ 293T cells but not CUL72/2 293T cells. Indicated plasmids were transfected to 293T cells or CUL7 knockout 293T cells. The protein levels were determined by Western blot. GAPDH was used as loading control. Graph shows relative AID-FLAG protein levels after different treatments. (F) FBXW11 (Figure legend continues) The Journal of Immunology 7

IgG3 and 25 mg/ml LPS, 5 ng/ml TGF-b (PeproTech), and 10 ng/ml anti- upregulated in the cells (Fig. 1A, 1B), suggesting that CRLs IgD (eBioscience) for induction of CSR to IgA. CH12F3 cells were treated participate in AID degradation. To further clarify which CUL li- with 5 ng/ml IL4, 0.2 mg/ml anti-CD40 (eBioscience), and 5 ng/ml TGF-b gases specifically degrade AID, various siRNAs for CUL1, CUL2, so that CSR would be induced to IgA. CUL3, CUL4A, CUL4B, CUL5, and CUL7, as well as a positive- Flow cytometry control REG-g, were respectively transfected into 293T cells m The cell surface markers were stained with fluorophore-conjugated Abs. expressing AID-NES -GFP. FACS analysis showed that only The following Abs were used in this analysis: FITC rat monoclonal anti- CUL7 and REG-g knockdown significantly upregulated the AID-GFP mouse IgG3 (clone R40-82, no. 553403; BD Biosciences), PE rat mono- median fluorescence intensity (Fig. 1C) and percentage of GFP-positive clonal anti-mouse IgG1 (clone A85-1, no. 550083; BD Biosciences), FITC cells (Supplemental Fig. 1), indicating that CUL7 E3 ligases specif- rat monoclonal anti-mouse IgA (clone C10-3, no. 559354; BD Biosciences), allophycocyanin rat monoclonal anti-mouse CD45R/B220 (clone RA3-6B2, ically mediated AID degradation. no. 561880; BD Biosciences), PE-Cy7 hamster monoclonal anti-mouse To confirm that CUL7 mediates AID degradation, 293T cells CD95 (clone Jo2, no. 557653; BD Biosciences), eFluor450 rat anti-mouse were cotransfected with siRNA for CUL7 and AID-FLAG– GL7 (clone GL-7, no. 4344710; eBioscience), Biotin conjunctive rat anti- expressing plasmids. Western blot analysis showed that CUL7 mouse AID (clone AID, no. 4331157; eBioscience), and PE-Cy5 streptavidin knockdown significantly enhanced AID levels (Fig. 2A). Addi- (no. 1938303; eBioscience). Flow cytometry was conducted on the LSR II (BD Biosciences). Data were analyzed with FlowJo software. tionally, CUL7 overexpression decreased AID levels, which was counteracted by MG132 (Fig. 2B). CUL7 knockdown by siRNA Real-time quantitative PCR decreased AID ubiquitination (Fig. 2C). In contrast, CUL7 RNA was isolated with TRIzol according to the manufacturer’s instructions overexpression increased AID ubiquitination but not H1464P-

(Ambion). The samples were treated with DNase (Promega) before reverse mutated CUL7, which impaired the ability to bind to RBX1 Downloaded from transcription. Total cellular mRNA was reverse transcribed using oligo dTs and assembly of polyubiquitin chains (31) (Fig. 2D). The Co-IP and random primers with the PrimeScript RT reagent Kit (TaKaRa). Real- time quantitative PCR was performed using gene-speciﬁc primers with assay indicated that CUL7 speciﬁcally bound to AID (Fig. 2E). SYBR premix Ex Taq Kit (Takara) on a CFX96 Real-Time system These results suggest that CUL7 E3 ligases bind to AID (Bio-Rad). b-Actin or HPRT mRNA was measured as endogenous controls. and promote AID degradation by mediating the ubiquitination CRISPR/Cas9-mediated CUL7 gene deletion of AID.

Dendra2 is a photoactivatable fluorescent protein (PAFP) that http://www.jimmunol.org/ The Cas9 gene was slightly modified to improve fidelity as described previously converts from green to red fluorescent states by exposure to 405-nm CUL7 (26). -specific single-guide RNA was cloned into LentiCRISPRv2.0 light (32). The Dendra2 red fluorescent state, which excludes plasmid (single-guide RNA: 59-GGTGCAGCTCTGTTCCGCGG-39). Lentiviruses were packaged in 293T cells through cotransfecting the newly synthesized protein, can be used to quantitatively LentiCRISPRv2.0, psPAX2, and pMD.2G plasmids. The 50% confluent measure the decay rate for a certain protein (32–34). After the 293T cells were infected with recombinant lentiviruses in the presence AID-Dendra2 fusion protein-expressing plasmid was transfected of 8 mg/ml polybrene. Single clones were selected by limited dilution into 293T cells, AID-Dendra2 red fluorescence decay was de- and maintained in medium with puromycin (1 mg/ml). CUL7-knockout in single clone 293T cells were verified by Western blotting and DNA tected following green-to-red conversion simulation by a 405-nm sequencing. surface light (Fig. 3A). Using this technique, we confirmed that CUL7 knockdown inhibited the decay of AID-Dendra2 and AID- by guest on September 24, 2021 Statistical analysis NESm-Dendra2 (Fig. 3B, 3C). In contrast, CUL7 overexpression Unless otherwise indicated, values are reported as the mean 6 SEM; values facilitated the decay of AID-Dendra2 and AID-NESm-Dendra2 of p ,0.05 were considered significant. Statistical significance between (Fig. 3D, 3E). As controls, CUL7 knockdown and overexpression two samples was determined by two-tailed Student t test. p values are did not influence the decay of fluorescent protein Dendra2 denoted in figures as *p , 0.05, **p , 0.01, and ***p , 0.001. (Fig. 3F, 3G). These results support that CUL7 E3 ligase influ- Results enced AID stability in cells. CUL7 functions as a specific E3 ligase for AID degradation CUL7 knockdown increases IgA class switching in CH12F3 APOBEC3G (A3G) belongs to the cytidine deaminase family, of B cells which AID is also a member, and induces hypermutation in newly To examine whether CUL7 influences B cell class switching by HIV-1 DNA to inhibit viral replication (27, 28). HIV-1 virion mediating AID degradation, mouse CUL7-specific siRNA was infectivity factor (Vif) connects A3G to the complex of CUL5 E3 transfected into CH12F3 cells. The knockdown efficiency of siRNA ligases, facilitating its degradation through the ubiquitin-proteasome in CH12F3 B cells was confirmed (Supplemental Fig. 2B). AID pathway (29). It is reasonable to assume that CRLs may also par- expression and class switching were detected after stimulation ticipate in the degradation of AID. Furthermore, it is well known that with anti-CD40, IL-4, and TGF-b (CIT) (35). The results showed the attachment of NEDD8 is necessary for CRL E3 ligase activity. that CUL7 knockdown enhanced AID protein levels in CH12F3 The NEDD8 inhibitor MLN4924 can inhibit the activity of CUL E3 cells and IgA class switching (Fig. 4A, 4B). Although CUL7 ligase and subsequently the degradation of Vif-mediated APOBEC3G knockdown did not influence AICDA mRNA transcription (30). For convenience, we examined the possible involvement (Fig. 4C) and IgH germline transcription (Fig. 4E, 4F), which of CUL E3 ligase in AID degradation by evaluating the inhibi- frequently occur with B cell class switching (36, 37), it increased m tory effect of MLN4924. When AID-NES -GFP– (AID with the levels of Ia-Cm circle transcripts (Fig. 4D), which reflect the mutated nuclear export signal) or AID-GFP–expressing plasmids frequency of IgA CSR induced by AID (38). These results suggest were transfected, 293T cells were treated with MLN4924 or the that CUL7 influences IgA class switching of CH12F3 cells by proteasome inhibitor MG132 and AID protein levels were directly regulating AID abundance.

knockdown increased IgA class switch of CH12F3 B cells. siNC or siFBXW11 were electrotransfected to CH12F3 B cells. After 12 h recovery, the transfected cells were stimulated with anti-CD40, IL-4, and TGF-b for 48 h. The IgA-positive cells were determined by ﬂow cytometry. Graph shows the percentage of IgA-positive cells. Except (B), all above data are representative of multiple experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 8 CUL7 REGULATES CSR BY UBIQUITINATING AND DEGRADING AID Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. CUL7-FBXW11 E3 ligase degraded AID through direct FBXW11-AID interaction and subsequent CUL7-FBXW11 complex assembly. (A) FBXW11 speciﬁcally interacted with AID. 293T cells were transfected with plasmids expressing AID-FLAG or GFP-FLAG. AID-FBXW11 interaction was examined by Co-IP with anti-FLAG beads followed by Western blotting with anti-FBXW11 Abs. GFP-FLAG as IP control; b-actin was used as a loading control. Graph shows normalized levels of AID-FLAG– or GFP-FLAG–interacted protein (B and C) Identiﬁcation of FBXW11 binding domain in AID. FBXW11-HA plasmids were cotransfected to 293T cells with plasmids expressing various deletions of AID-GFP (Figure legend continues) The Journal of Immunology 9

CUL7-FBXW11 complex mediates AID degradation FBXW11 bound to AID and was required for CUL7-mediated F-box proteins are components of SCF ubiquitin-ligase complexes degradation of AID. We predicted that CUL7 assembles with that contain Skp1, CUL1, or CUL7, and an F-box protein (39). FBXW11 in the presence of AID. To evaluate this hypothesis, we cotransfected CUL7 with plasmids expressing AID or These proteins directly bind to their substrates to induce ubiquitin- H56R/E58Q mediated degradation. F-box proteins are classified into three AID (AID with no cytidine deaminase activity) (43). families: FBXL containing an F-box and leucine-rich repeats, Indeed, the interaction of CUL7 and FBXW11 was weak in the absence of AID or AIDH56R/E58Q overexpression. Interestingly, FBXW containing an F-box and WD tryptophan-aspartic acid H56R/E58Q repeats, and FBXO containing F-box with or without another overexpression of AID or AID followed by etoposide, motif (40). CUL7 generally assembles with the F-box protein a DNA damage inducer, treatment significantly promoted the FBXW8 to form a complex and functions as an E3 ligase (41). To FBXW11-CUL7 interaction (Fig. 6D). These data suggest that determine whether the influence of CUL7 on AID degradation interaction of CUL7-FBXW11 was dependent on AID deaminase activity and its induced DNA damage response. In addition, we depends on FBXW8, we knocked down FBXW8 with siRNA and H56R/E58Q simultaneously overexpressed CUL7 in 293T cells. FBXW8 found that AID showed weak interaction with FBXW11- knockdown by siRNA in 293T cells was found to be efficient CUL7 but was rescued by following etoposide treatment (Fig. 6E). (Supplemental Fig. 2C). However, FBXW8 knockdown did not It suggests that the interaction of AID and E3 ligases was also inhibit CUL7-mediated AID degradation (Fig. 5A). We predicted dependent on AID-induced DNA damage response. Moreover, a Co-IP assay showed that FBXW11 interacted with AID not only in that other members of the FBXW family participate in CUL7- +/+ +/+ mediated AID degradation. Using siRNA screening for mem- CUL7 293T cells but also in CUL7 293T cells (Fig. 6F). bers of the FBXW family, we found that FBXW11 knockdown In contrast, when FBXW11 was knocked down with siRNA in Downloaded from significantly inhibited AID degradation mediated by CUL7 293T cells, the CUL7-AID interaction was significantly decreased overexpression (Fig. 5B). Western blot analysis confirmed that (Fig. 6G). These data suggest that AID contacts CUL7-FBXW11 when FBXW11 was knocked down, CUL7 overexpression did E3 ligase by directly binding to FBXW11 but not to CUL7. Thus, not promote AID degradation (Fig. 5C). Moreover, transfection CUL7-FBXW11 E3 ligase may specifically mediate the degra- of FBXW11-specific siRNA without CUL7 overexpression en- dation of AID. http://www.jimmunol.org/ hanced AID protein levels (Fig. 5D). Alternatively, to confirm fl/fl + whether FBXW11-mediated AID degradation depends on CUL7, CUL7 CD19-cre mice B cells show high levels of AID and we constructed a CUL7 knockout 293T cell line using CRISPR- IgA, IgG1 class switching Cas9. Frameshift mutations in CUL7 and deficiency of CUL7 To examine whether CUL7 knockdown influenced AID expression protein were confirmed (Supplemental Fig. 2E). FBXW11 in vivo, we immunized CUL7 conditional knockout mice (44) with wasoverexpressedinCUL7knockout293Tcells(CUL72/2 NP-CGG. We first confirmed the protein expressions of CUL7 293T cells) and wild-type 293T cells (CUL7+/+ 293T cells). We and FBXW11 in germinal center B cells (B220+GL7+CD95+) found that FBXW11 overexpression mediated AID degradation in of CUL7fl/flCD19-cre+ or CUL7fl/flCD19-cre2 mice by Western CUL7+/+ 293T cells but not in CUL72/2 293T cells. However, blotting (Fig. 7A). The protein levels of AID in germinal center by guest on September 24, 2021 when CUL7 was rescued in CUL72/2 293T cells, FBXW11 ef- B cells (B220+GL7+CD95+) were then examined by FACS. The ficiently mediated AID degradation (Fig. 5E). Moreover, IgA class results showed that AID levels were increased in CUL7fl/flCD19-cre+ switching in CH12F3 cells transfected with FBXW11-specific mice compared with in littermate CUL7fl/flCD19-cre2 mice (Fig. 7B). siRNA was significantly increased (Fig. 5F). The efficiency of To determine whether CUL7 knockout in B cells influenced class mouse FBXW11 knockdown by siRNA in CH12F3 B cells was switching, CD43-negative naive B cells in the spleen of mice were confirmed (Supplemental Fig. 2D). sorted and stimulated with various cytokines, including IL-4 and LPS To examine whether FBXW11 directly interacts with AID, for IgG1 class switching; LPS only for IgG3 class switching; and we transfected AID-FLAG–expressing plasmids into 293T cells. LPS, TGF-b, and anti-IgD for IgA class switching. The results An immunoprecipitation (IP) assay showed that AID specifically showed that IgG1 and IgA class switching increased in naive B cells bound to FBXW11 (Fig. 6A). Plasmids expressing various after stimulation of CUL7fl/flCD19-cre+ mice compared with in deletions of AID and FBXW11 were transfected into 293T cells CUL7fl/flCD19-cre2 mice (Fig. 7C, 7D). However, IgG3 class switch followed by a Co-IP assay, and we further found that the binding elevation did not occur in B cells from CUL7fl/flCD19-cre+ mice domain of FBXW11 for AID was at its 102–151 aa (Fig. 6B, 6C). (Fig. 7E). These results suggest that CUL7 conditional knockout in However, a previous study showed that CUL7 bound to FBXW8 B cells increases AID expression in B cells in vivo, subsequently but not FBXW11 in 293T cells (42). Our data showed that promoting IgG1 and IgA class switching.

fusion protein. Their interactions were examined by Co-IP with anti-HA beads followed by Western blotting with anti-GFP Abs. GAPDH was used as a loading control. Graph shows normalized levels of FBXW11-interacted AID-GFP protein. (D) FBXW11 interacted with CUL7 was dependent on AID deaminase activity. CUL7-HA plasmids were cotransfected to 293T cells with the empty vector or plasmids expressing AID or AIDH56R/E58Q.TheAIDH56R/E58Q transfected cells were treated with Etoposide for 24 h. CUL7-FBXW11 interaction was examined by Co-IP with anti-HA beads followed by Western blotting with anti-FBXW11 Abs. Graph shows relative normalized levels of CUL7-interacted FBXW11 after different treatments. (E) FBXW11-CUL7-AID interaction was dependent on AID deaminase activity. 293T cells were transfected with AID or AIDH56R/E58Q.TheAIDH56R/E58Q-transfected cells were treated with Etoposide for 24 h. AID-CUL7-FBXW11 interaction was examined by Co-IP with anti-FLAG beads followed by Western blotting with anti-CUL7 and anti-FBXW11 Abs. Graph shows relative normalized levels of AID-interacted FBXW11 and CUL7 after different treatments. (F) FBXW11-AID interaction does not rely on CUL7. AID-FLAG plasmids were transfected to CUL7+/+ or CUL72/2 293T cells. FBXW11-AID interaction was examined by Co-IP with anti-FLAG beads followed by Western blotting with anti-FBXW11 Abs. GAPDH was used as a loading control. Graph shows normalized levels of AID-interacted FBXW11 in different cells. (G) FBXW11 is required for the CUL7-AID interaction. AID-FLAG plasmids were cotransfected with siFBXW11 or siNC to 293T cells. CUL7-AID interaction was examined by Co-IP with anti-FLAG beads followed by Western blotting with anti-CUL7 Abs. GAPDH was used as a loading control. Graph shows normalized levels of AID-interacted CUL7 after different treatments. All above data are representative of multiple experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 10 CUL7 REGULATES CSR BY UBIQUITINATING AND DEGRADING AID Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. Cul7fl/flCD19-cre+ mice exerted high levels of AID and high frequency of IgA, IgG1 class switch. (A) CUL7 and FBXW11 protein levels in sorted germinal center B cells (B220+GL7+CD95+)ofCUL7fl/flCD19-CRE+ or CUL7fl/flCD19-CRE2 mice; GAPDH was used as a loading control. Graph shows normalized protein levels of CUL7 and FBXW11 in sorted cells with different genotype. (B) Representative flow cytometry of AID protein levels in germinal center B cells (B220+GL7+CD95+)ofCUL7fl/flCD19-CRE+ or CUL7fl/flCD19-CRE2 mice (n = 6). (C–E) Representative flow cytometry of CSR to IgG1 (C), IgA (D), and IgG3 (E) in splenic naive B cells in CUL7fl/flCD19-Cre+ or CUL7fl/flCD19-Cre2 mice. Data (n = 4) are represented as the mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 11

Discussion enhancement. Conversely, CSR is a deletion recombination Although AID is a nuclear cytoplasm-shuttling protein, it is de- mechanism that replaces Cm, the first CH gene expressed during graded mainly in the nucleus (16). AID protein containing mu- B cell development, with the downstream CH gene. CSR typically tations in the exporting signal (mutNES-AID) was sequestered in occurs in long and repetitive switch (S) regions upstream of the the nucleus and showed an 8-fold shorter t1/2 (2.5 versus 20 h) various IgH region exons (36). A positive correlation between the than did AID with mutations in the import signal (mutNLS-AID) S region length and CSR frequency was confirmed previously (16). In contrast, it has been also reported that the Hsc70- (59). The Sg1 region for IgG1 class switching (10 kb) and Sa associated E3 ubiquitin ligase CHIP can destabilize AID in the region for IgA class switching (4.2 kb) are larger than those of cytoplasm (10). However, CHIP is regarded as a quality control E3 Sε (1 kb), Sg3 (2.5 kb), Sg2b (5.0 kb), and Sg2a (2.5 kb) (60–64). ligase that selectively ubiquitylates unfolded or misfolded proteins Alternatively, it is possible that long Sg1 and Sa regions provide by working with heat shock protein chaperones (45). Although more targets for AID and therefore are more sensitive to AID ubiquitin E3 ligase MDM2 and RNF126 interact with AID, no upregulation. studies have demonstrated that they participate in AID degrada- As reported in the International Mouse Phenotyping Consortium tion (46, 47). Moreover, the transcription factor YY1 physically databank, CUL7 heterozygous mice showed a lower percentage of CD19+ B cells but with normal CD3+ T and CD8+ T cells as well interacts with and destabilizes AID in the nucleus by decreasing + AID ubiquitination (48, 49). as a slight elevation of CD4 T cells (Supplemental Fig. 3B, 3E). In this study, we showed that CUL7-FBXW11 mediated the Combined with our findings and other reports showing that AID ubiquitination of AID and its subsequent degradation. In contrast expressed in immature B cells mediated B cell apoptosis (65), we to a previous study reporting that overexpressed Myc3-CUL7 lo- predict that CUL7 also regulates AID levels in immature B cells. Downloaded from calizes mainly in the cytoplasm (50), we found that endogenous More specifically, CUL7 knockout may lead to high levels of AID in immature B cells and induce B cell apoptosis, resulting in a low CUL7 has no specific cytoplasmic distribution preference in an + immunofluorescence assay using an anti-CUL7 Ab (Supplemental CD19 B cell percentage. The germinal center is a highly dynamic microenvironment in Fig. 2A). As CUL7 overexpression promoted AID degradation which B cells undergo further development and maturation and both in the cytoplasm and nucleus, CUL7 may regulate the AID

various Abs are generated and maturated. Germinal center B cells http://www.jimmunol.org/ protein level either in the nucleus or cytoplasm to maintain a stock shuttle between the light zone where high-affinity Abs are posi- quantity of AID. tively selected and dark zone where highly expressed AID engraves FBXW11 (b-TrCP2) and its homolog FBXW1 (b-TrCP1) were IgH genes in B cells (66). The CUL7-FBXW11 ubiquitination originally found to bind to HIV-1 Vpu and participate in the system, possibly in combination with the REG-g nonubiquitination degradation of CD4 or the antiviral factor Tetherin/BST-2 (51, 52). system, may mediate the rapid degradation of AID in light zone Both b-TrCP1 and b-TrCP2 also mediate the degradation of B cells to guarantee normal affinity maturation and isotype class CDC25A, inhibiting CDK1 activity and subsequently arresting the switch. However, we found no significant differences in CUL7, cell cycle before DNA repair. This step is important during the FBXW11,andREG-g mRNA expression between light zone and

genotoxic stress response (53, 54). FBXW11 typically cooperates by guest on September 24, 2021 dark zone B cells by analyzing the public array data (GSE38712) with CUL1 to form an SCF ubiquitin E3 ligase (55). We found (Supplemental Fig. 4). This suggests that posttranslation modifica- that FBXW11 interacts with CUL7 in an AID-dependent manner. tions of CUL7, FBXW11, or REG-g proteins, but not transcription A previous study indicated that CUL7-CUL1-FBXW8 formed regulation, directly regulate their activities to timely and quickly heterodimeric E3 ligases and broadened the field of substrate regulate AID abundance in B cells during shuttling between the light specificity (56). We initially considered that CUL7-FBXW8- and dark zones. Identification of new signaling pathways, particularly CUL1-FBXW11 forms a large complex and functions as an E3 posttranslational modification signaling pathways of CUL7-FBXW11 ligase to mediate the degradation of AID. This possibility was or REG-g to regulate AID stability, will provide insight into the excluded, as CUL1 and FBXW8 knockdown did not impact AID process of Ab diversity generation and vaccine development. degradation (Figs. 1C, 5A). In this study, FBXW11 was assembled with CUL7 and functioned as an E3 ligase to mediate the deg- Acknowledgments radation of AID. Although this process depends on the deami- We thank Dr. Tasuku Honjo (Kyoto University, Kyoto, Japan) for CH12F3 nase activity of AID, the various cellular signals activated by cells through Dr. Hu Wenjun (Kyoto University, Kyoto, Japan) and Dr. Meng AID-induced DNA damage response may also facilitate uncon- Feilong (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, ventional CUL7-FBXW11 interaction and AID-FBXW11 inter- China). action (57, 58). In CUL7 conditional knockout mice, we found that the AID level Disclosures was increased in germinal center B cells. However, elevated The authors have no financial conflicts of interest. AID only increased IgG1 and IgA class switching without IgG3 isotypes. To determine whether the different expression of AID after various cytokine treatment leads to such results, we detected References AID protein levels of CD432 naive B cells in CUL7fl/flCD19- 1.Muramatsu,M.,K.Kinoshita,S.Fagarasan,S.Yamada,Y.Shinkai,and + fl/fl 2 T. Honjo. 2000. Class switch recombination and hypermutation require CRE or CUL7 CD19-CRE mice after stimulation with various activation-induced cytidine deaminase (AID), a potential RNA editing en- reagents for 72 h. We found that a higher AID protein level indeed zyme. Cell 102: 553–563. 2. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, occurred when naive B cells were stimulated to IgG1 or IgA CSR M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al. 2000. Activation- compared with IgG3 CSR (Supplemental Fig. 3A). However, induced cytidine deaminase (AID) deficiency causes the autosomal recessive CUL7 knockout increased AID protein levels when stimulating to form of the Hyper-IgM syndrome (HIGM2). Cell 102: 565–575. 3. Robbiani, D. F., A. Bothmer, E. Callen, B. Reina-San-Martin, Y. Dorsett, IgG1, IgA, and IgG3 CSR (Supplemental Fig. 3A). These data S. Difilippantonio, D. J. Bolland, H. T. Chen, A. E. Corcoran, A. Nussenzweig, indicated that, although the CUL7 knockout upregulated AID in and M. C. Nussenzweig. 2008. AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations. Cell 135: 1028–1038. the condition for inducing IgG3, it did not upregulate IgG3 CSR 4. Ramiro, A. R., M. Jankovic, E. Callen, S. Difilippantonio, H. T. Chen, frequency partially because of the relatively lower AID expression K. M. McBride, T. R. Eisenreich, J. Chen, R. A. Dickins, S. W. Lowe, et al. 12 CUL7 REGULATES CSR BY UBIQUITINATING AND DEGRADING AID

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siNC siCUL1 siCUL2

46.2% 47.2% 46.9%

siCUL3 siCUL4A siCUL4B

50.1% 53.1% 51.6% FSC

siCUL5 siCUL7 siREG!

46.8% 62.0% 61.9%

GFP signal

Supplemental Figure 1 siRNA screening for CUL Family to affect AID protein levels. Individual siRNA for CULs and a plasmid expressing AID-NESmu-GFP (F193A, L196A) were co-transfected to 293T cells. Forty-eight hours post transfection, the cells were digested by Trypsin-EDTA and resuspended in PBS followed FACS analysis. Supplemental Figure 2 A. anti-CUL7 DAPI Merge

Flied1

Flied2

B. C.

*** *** *** *** Relative FBXW8 mRNA expression Relative mCUL7 mRNA expression siNC simCUL7-1 simCUL7-2 siNC siFBXW8-1 siFBXW8-2 D. E.

*** ***

1 2 CUL7 1:WT_293T

GAPDH 2:CUL7_KO_293T

Relative mFBXW1 mRNA expression siNC simFBXW11-1 simFBXW11-2

Supplemental Figure 2 (A) Localization of CUL7 in 293T cells. Fixed 293T cells were stained with anti-CUL7 antibody and DAPI. The Fluorescence-flied micrograph images were obtained and processed in the same way. The bar indicated 50 mm. (B) Knockdown efficiency of mouse CUL7-specific siRNA in CH12F3 cells. (C) Knockdown efficiency of human FBXW8-specific siRNA in 293T cells. (D) Knockdown efficiency of mouse FBXW11-specific siRNA in CH12F3 cells. (E) Frame shift mutation of CUL7 gene in CUL7-deletion 293T cells mediated by CRISPR-Cas9. Supplemental Figure 3 A. CSR IgA *** for *** CSR IgD - *

IgG1 CSR

b+anti levels for - *** 4 IgG3 -

stimulation for

No LPS+IL LPS+TGF LPS Protein ** Cre- Cre+ Cre- Cre+ Cre- Cre+ Cre- Cre+ AID AID

Actin Relative Cre- Cre+ Cre- Cre+ Cre- Cre+ Cre- Cre+

No LPS+ LPS+ LPS for stimulation IL-4 for TGF-b+ IgG3 CSR IgG1 CSR anti-IgD for B. C. IgA CSR

** *

D. E.

ns ns

DataB to E came from IMPC(https://www.mousephenotype.org/data/experiments?geneAccession=MGI:1913765) and organized by authors Supplemental Figure 3 CUL7 knockout increased AID protein levels when stimulating to IgG1, IgA, and IgG3 CSR. The AID protein levels of cytokine stimulated resting B cells in CUL7fl/flCD19-CRE+ or CUL7fl/flCD19-CRE- mice were determined by western blot using anti-AID. Beta-actin was used as a loading control. Graph shows relative AID protein levels in B cells with different genotype after different treatments(A). CUL7 KO heterozygote mice showed decreased CD19+ B cells (B), increased faintly CD4+ T cells (C), but normal CD8+ T cells (D), CD3+ T cells (E). All data came from International Mouse Phenotyping Consortium (IMPC) and organized by us. Supplemental Figure 4 A. B.

Mouse FBXW11 expression Human FBXW11 expression ns ns

C. D. Mouse CUL7 expression Human CUL7 expression ns ns

E. Mouse PSME3 (REG-g) expression F. Human PSME3 (REG-g) expression

ns *

Supplemental Figure 4 No significant difference of CUL7, FBXW11 and REG-g mRNA expression between light zone and dark zone B cells by analyzing public array data (GSE38712).