CUL7 E3 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 October 2, 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

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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 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 . The ac- tivity and levels of AID are tightly controlled by various mechanisms. In this study, we found that CUL7 E3 ubiquitin ligases

specifically mediated AID ubiquitination. CUL7 overexpression or knockdown influenced the decay of AID, affecting AID protein Downloaded from 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

potential target for developing immunologic adjuvants for vaccines of various pathogens such as HIV-1 and influenza viruses. http://www.jimmunol.org/ 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 , 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- depend on the activity of activation-induced cytidine deaminase clear AID is exported to the cytoplasm by CRM1 (13) or quickly by guest on October 2, 2021 (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, ; 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 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 to CUL proteins is required for CRL ubiquitin ligase activity (Kyoto University) and Dr. F. Meng (Shanghai Institue of Biochemistry Downloaded from (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

CUL7 in vitro and in vivo increased the protein levels of AID and subjected to Western blot. For coimmunoprecipitation (Co-IP), the http://www.jimmunol.org/ 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 October 2, 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 October 2, 2021

FIGURE 2. CUL7 knockdown or overexpression influenced protein levels and ubiquitination of AID in 293T cells. (A) CUL7 knockdown upreg- ulated 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 October 2, 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 October 2, 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 we