Cutting Edge: The Transcription Factor Sox2 Regulates AID Expression in Class-Switched B Cells This information is current as Lauren J. DiMenna, Wei-Feng Yen, Laura Nicolas, Rahul of September 25, 2021. Sharma, Zara N. Saldanha and Jayanta Chaudhuri J Immunol 2017; 198:2244-2248; Prepublished online 10 February 2017; doi: 10.4049/jimmunol.1502266 http://www.jimmunol.org/content/198/6/2244 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/02/09/jimmunol.150226 Material 6.DCSupplemental http://www.jimmunol.org/ References This article cites 35 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/198/6/2244.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology Cutting Edge: The Transcription Factor Sox2 Regulates AID Expression in Class-Switched B Cells Lauren J. DiMenna,*,† Wei-Feng Yen,* Laura Nicolas,* Rahul Sharma,* Zara N. Saldanha,* and Jayanta Chaudhuri*,† IgH class switch recombination (CSR) occurs through during CSR, off-target activity of AID within B cells can the deliberate introduction of activation-induced cyti- generate translocations commonly seen in the development of dine deaminase (AID)-instigated DNA double-strand lymphomas (1, 3). Therefore, AID expression is tightly reg- breaks into the IgH loci. Because double-strand breaks ulated at multiple levels, and its transcription alone is con- are generally highly toxic, mechanisms that regulate trolled by at least a dozen transcription factors (4). However, AID expression are of much relevance to CSR and geno- the mechanism that turns off AID in B cells that have already mic integrity; however, effectors of such regulatory initiated and/or completed CSR is poorly understood. Downloaded from processes are still poorly understood. In this article, we The transcription factor sex determining region Y-box 2 show that the transcription factor sex determining region (Sox2) is primarily known for its role in early development, Y-box 2 (Sox2) is expressed in activated B cells, but al- where it can be found in embryonic stem cells, the differen- most exclusively in those that have undergone CSR. We tiating eye and gut, and in primordial germ cells (5–7). It also was detected in some adult stem cell populations and skin demonstrate that enforced expression of Sox2 in splenic http://www.jimmunol.org/ cells (8, 9). More recently, Sox2 expression was demonstrated B cells severely inhibits AID expression and CSR, whereas in neutrophils, where it was shown to play a noncanonical deletion of Sox2 increases the frequency of IgH:c-Myc role as a pathogen sensor in the cytoplasm (10). Finally, Sox2 translocations. These results suggest that Sox2 may is also one of the four transcription factors (with Klf4, Oct4, regulate AID expression in class-switched B cells to and c-Myc) that are vital for reprogramming somatic cells suppress genomic instability associated with CSR. into induced pluripotent stem cells (11). The Journal of Immunology, 2017, 198: 2244–2248. In this article, we report that Sox2 is expressed in class- switched B cells and that its enforced expression led to a ature IgM-expressing B cells in peripheral lym- drastic reduction in levels of AID and frequency of CSR. by guest on September 25, 2021 phoid organs, such as the spleen, lymph nodes, and Conversely, deletion of Sox2 in mature B cells increased AID M Peyer’s patches, are activated upon Ag encounter expression and the frequency of IgH:c-Myc translocations. and undergo class switch recombination (CSR), a deletional- Thus, Sox2 appears to be a novel factor that turns off critical recombination reaction that replaces the Cm C region seg- aspects of the CSR program in class-switched B cells to e ment with a set of downstream CH genes (Cg,C ,orCa) (1, maintain genomic integrity. 2). The B cell thereby changes from expressing IgM to one expressing IgG, IgE, or IgA, with each secondary isotype Materials and Methods having a distinct effector function during an immune re- Mice, B cell cultures, and retroviral transduction sponse. CSR occurs between repetitive DNA elements termed Wild-type C57BL/6J mice were obtained from the Jackson Laboratory, and 2/2 switch (S) regions that precede each CH segment and requires AID mice were a gift from T. Honjo (12). Primary B cells were isolated from the spleen, stimulated with anti-CD40 (1 mg/ml; HM40-3; eBioscience) germline transcription through the CH genes and activation- and IL-4 (12.5 mg/ml; 404-ML; R&D Systems), LPS (10 mg/ml; Sigma), or induced cytidine deaminase (AID) (1, 2). Current models LPS+IL-4, and analyzed for CSR, as described previously (13). Sox2 condi- posit that AID-mediated deamination of transcribed S regions tional mice (Sox2flox) were generated by Shaham et al. (14) and purchased instigates generation of DNA double-strand breaks (DSBs), from the Jackson Laboratory (http://www.informatics.jax.org/reference/J: and end-joining of DSBs between donor (usually Sm) and 152850). Splenic B cells were transduced with retroviral vectors pMIR or e pMIR-Sox2 using spinfection, as described earlier (13). Immunization with downstream acceptor S regions (Sg,S,orSa) completes NP-CGG and analysis of germinal center (GC) B cells and NP-specific re- CSR (1, 2). Although S regions are primary targets of AID sponse by flow cytometry and ELISA were performed as described earlier *Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10021; The RNA sequencing data presented in this article have been submitted to the National and †Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi. of Medical Sciences, New York, NY 10065 nlm.nih.gov/geo/query/acc.cgi?acc=GSE93131) under accession number GSE93131. ORCIDs: 0000-0003-1057-3729 (Z.N.S.); 0000-0002-3838-0075 (J.C.). Address correspondence and reprint requests to Dr. Jayanta Chaudhuri, Memorial Sloan Kettering Cancer Center, New York, NY 10065. E-mail address: [email protected] Received for publication October 22, 2015. Accepted for publication January 23, 2017. The online version of this article contains supplemental material. This work was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases (Grants 1R01AI072194 and 1R01AI124186), a Abbreviations used in this article: AID, activation-induced cytidine deaminase; CSR, National Institutes of Health/National Cancer Institute Cancer Center Support class switch recombination; DSB, double-strand break; GC, germinal center; RNA-Seq, Grant (P30 CA008748), the Memorial Sloan Kettering Cancer Center Functional Geno- RNA sequencing; S, switch; Sox2, sex determining region Y-box 2. mic Institute, and the Starr Cancer Research Foundation (Grant I7-A767) (to J.C.) and National Institutes of Health Grant 5T32 AI007621 (to L.J.D.). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502266 The Journal of Immunology 2245 (15). To purify GC B cells, splenocytes from immunized mice were stained 2 2 2 in splenic B cells activated to undergo CSR to IgG1 ex vivo with B220, GL7, and Fas, and B220+GL7+Fas+ (GC+) or B220+GL7 Fas (GC ) + + with anti-CD40 and IL-4 (Fig. 1A). Interestingly, when ac- cells were collected by passing through a cell sorter. IgM and IgG1 Bcellswere + + purified by cell sorting following stimulation of splenic B cells with anti-CD40 + tivated B cells were sorted into IgM and IgG1 populations IL-4 for 4 d. (Supplemental Fig. 1A), Sox2 protein was found to be more abundant in the class-switched IgG1+ fraction (Fig. 1B). Western blotting and cell-division assays Additionally, Sox2 expression in AID-deficient activated Whole-cell extracts were prepared in RIPA lysis buffer (13). Primary Abs were B cells was markedly lower than in wild-type B cells (Fig. 1C). as follows: anti-AID (16), anti-Sox2 (R&D Systems), anti-Gapdh (6C5; Millipore), anti-tubulin (T9026; Sigma), anti-Batf (D7C5; Cell Signaling The delayed kinetics of Sox2 expression and its abundance in Technology), anti-p21 (B-2), anti-cyclinD2 (M-20), and anti-Irf4 (M-17) (all class-switched B cells prompted us to query the effect of from Santa Cruz Biotechnology). Uninfected and retrovirally infected cells enforced Sox2 expression on CSR. Mouse splenic B cells were were stained with the CellTrace CFSE Cell Proliferation Kit (Molecular retrovirally infected with empty vector pMIR (expressing Probes) immediately following the first spinfection (or at 24 h of stimulation for control cells), according to the manufacturer’s instructions. Staining was mCherry marker) or pMIR-Sox2, and CSR to IgG1 was detected by flow cytometry at 0 and 48 h. assessed in transduced B cells. Overexpression of Sox2 led to a reproducible and marked reduction in CSR to IgG1 (Fig. 1D, Quantitative real-time PCR 1E, Supplemental Fig. 1B). Total RNA was extracted using TRIzol Reagent, according to the manufac- Consistent with the role of Sox2 in promoting cellular turer’s instructions, followed by cDNA synthesis of 1 mg of total RNA with a qScript cDNA Synthesis Kit (Quantabio).