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). iQ SYBR Green Supermix (Bio- proliferation (19), enforced expression of Sox2 in B cells led to Rad) was used for quantification of mRNA, which was performed on a Bio- decreased levels of the cell cycle inhibitor p21 and increased Downloaded from Rad CFX96 Real-Time PCR Detection system. CFX Manager software was levels of cyclin D2, the major cyclin involved in B cell prolif- used to determine the crossing points for incorporation of the SYBR Green. eration (Supplemental Fig. 1C) (20, 21). Furthermore, dilution Relative expression was determined with b-actin as a reference gene. Primers are as follows: Batf 59-AGCTTCAGCCGCTCTCCT-39 and 59-GGTGT- of CFSE, a surface staining dye, showed that Sox2-expressing CGGCTTTCTGTGTCT-39,Irf459-ACGCTGCCCTCTTCAAGG-39 and cells proliferated slightly better than did control cells 59-GCTCCTCTCGACCAATTCCT-39,p2159-TCCACAGCGATATCC- (Supplemental Fig. 1D); however, CSR was markedly im- AGACA-39 and 59-GCGCAACTGCTCACTGTC-39, CyclinD2 59-GCT- paired, regardless of the extent of proliferation (Supplemental http://www.jimmunol.org/ AGGAACATGCACACTGC-39 and 59-CTGGGGCTTCACAGAGTTGT- 39, g1-GLT: 59-GGCCCTTCCAGATCTTTGAG-39 and 59-GGATCCA- Fig. 1D). Thus, impaired CSR in Sox2-expresing cells was not GAGTTCCAGGTCACT-39, g3-GLT: 59-AACTACTGCTACCACCAC- due to a defect in cell proliferation. CACCAG-39 and 59-ACCAAGGGATAGACAGATGGGG-39,andm-GLT: 59-CTCTGGCCCTGCTTATTGTTG-39 and 59-GAAGACATTTGGGAA- GGACTGACT-39.PrimersforAID,b-actin, m-chain, g1-chain, and g3were described previously (17).

RNA sequencing A paired-end RNA sequencing (RNA-Seq) library was prepared as described, by guest on September 25, 2021 and sequencing was performed on an Illumina HiSeq2000 with 30 3 106 reads per sample. Determination of differential expression was based on a minimum of a 2-fold change in expression and $ 200 average reads in the higher sample. Gene ontology analysis was carried out using the Database for Annotation, Visualization and Integrated Discovery. The heat maps were generated using R software packages. The RNA-Seq data were submitted to the National Center for Biotechnology Information Gene Expression Om- nibus (accession number GSE93131).

Translocation assay Translocations between IgH and c-Myc were assessed by PCR using primers and PCR conditions described previously (18). Briefly, genomic DNA from ∼1 3 105 cells was subjected to two rounds of amplification using nested primers and the Expand Long Template PCR system (Roche); products were resolved on agarose gel and probed by Southern blotting using IgH and c-Myc probes, as previously described (18). Statistical analysis Statistical significance was assessed using a Student t test. FIGURE 1. Enforced Sox2 expression alters CSR and AID levels. (A) Immunoblot of protein extracts from wild-type splenic B cells stimulated with Results and Discussion anti-CD40 and IL-4 for the indicated time and probed for expression of Enforced Sox2 expression severely impairs CSR Sox2, AID, or Gapdh (loading control) (n = 4). (B) Representative immu- noblot of purified IgM+ or IgG1+ splenic B cells obtained from anti-CD40 Although CSR is essential for B cell–mediated immunity, it and IL-4 cultures; tubulin, loading control (n = 3). (C) Representative im- 2 2 proceeds through the deliberate generation of DSBs into the munoblot of activated splenic B cells from wild-type or AID / mice (n = 3). B cell genome (2). Therefore, it is critical that the process is (D) Representative flow cytometry of B cells infected with pMIR-Sox2 or finely regulated, not only for proper control of CSR, but also pMIR control vector. pMIR expresses mCherry from an IRES element + for the maintenance of genomic integrity. However, factors downstream of the test gene. Cells were gated first on the mCherry pop- + + that regulate AID expression once CSR has been initiated are ulation as a marker for infection and then on B220 IgG1 cells. Bar graph shows CSR of 15 independent experiments; CSR in the pMIR control poorly defined. In our effort to understand the process by population was set at 100%. (E) Immunoblot shows expression of Sox2, AID, which the combination of the four transcription factors (Sox2, and tubulin (loading control). (F) mCherry+ B cells were sorted 72 h post- c-Myc, Klf4, and Oct4) converts a B cell into an induced infection and analyzed for Aicda expression by quantitative PCR; Aicda level pluripotent stem cell, we discovered that Sox2 was expressed in pMIR sample was set to 1 (n = 3). ***p , 0.001. 2246 CUTTING EDGE: Sox2 REGULATES AID EXPRESSION

AID is induced by CSR stimuli in B cells and is absolutely required for CSR (12, 22, 23). Therefore, we assessed AID levels in B cells transduced with pMIR-Sox2. Sox2 overexpression led toadrasticreductioninAIDprotein(Fig.1E)andAicda (encoding AID) mRNA (Fig. 1F). The effect of Sox2 expression on AID levels was not limited to cells stimulated to undergo CSR to IgG1. Overexpression of Sox2 in splenic B cells stimulated with LPS also impaired CSR and AID expression (Supplemental Fig. 1E, 1F). Thus, reduction in AID levels could account for the impaired CSR observed in B cells overexpressing Sox2.

Sox2 targets known regulators of AID expression To examine whether restoring AID levels could rescue CSR, mouse splenic B cells were coinfected with pMIR-Sox2 and pMIG-AID or control vectors. Despite comparable levels of AID in pMIR/pMIG-AID and pMIR-Sox2/pMIG-AID coinfected samples, CSR to IgG1 was not significantly re- stored (Fig. 2A–C), suggesting that Sox2 could be impacting Downloaded from multiple parameters affecting CSR. Indeed, germline tran- scription from Ig1 was markedly impaired, whereas that through Im, which is constitutively transcribed, was unaltered (Fig. 2D). A similar reduction in germline transcription from Ig3 was observed in Sox2-overexpressing cells stimulated with LPS (Supplemental Fig. 1G). As expected, exogenous AID http://www.jimmunol.org/

FIGURE 3. Sox2 dampens expression of the AID regulators Batf and Irf4. A Cells were sorted based on mCherry expression as a marker of infection. ( ) by guest on September 25, 2021 RNA-Seq was performed on samples from three independent infections. Differential expression of genes involved in CSR and in regulating AID ex- pression (upper panel) or genes expressed during B cell activation and dif- ferentiation (lower panel) are shown. Genes shown below the arrow were not significantly different in the Sox2-expressing cells. Quantitative PCR and immunoblot analysis of Irf4 (B) and Batf (C) expression levels in infected cells (n = 3). *p , 0.05, **p , 0.01.

expression did not restore levels of germline transcripts (Supplemental Fig. 1H). Therefore, it appears that Sox2 regulates transcription of induced promoters in activated B cells and that the CSR defect observed in Sox2-expressing B cells is likely due to defects in induced germline tran- scription and AID expression. To examine the global effects of Sox2 overexpression, we purified pMIR- and pMIR-Sox2–transduced B cells based on mCherry expression and subjected them to RNA-Seq analysis. Sox2 overexpression led to a significant increase in 702 genes and a decrease in 388 genes (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE93131). These genes were grouped FIGURE 2. Exogenous AID expression cannot restore CSR in Sox2- into putative functional categories using the Database for overexpressing cells. (A) Representative flow cytometry of splenic B cells Annotation, Visualization and Integrated Discovery. Impor- coinfected with pMIR, pMIR-Sox2 (Sox2), pMIG, and pMIG-AID (AID). tantly, although Sox2 overexpression led to an increase in the mCherry from pMIR and GFP from pMIG-based vectors served as surrogates expression of many genes involved in developmental path- for infection. (B) Average CSR in coinfected cells, with CSR in pMIR/pMIG- ways, the master regulators of B cell identity (including Pax5, C coinfected cells set at 100% (n = 3). ( ) Representative immunoblot showing E2A, and Bcl6) (24–26) were unaltered (Fig. 3A). Consistent expression of exogenous Sox2 and AID in the experiment from (A). (D) IgM and IgG1 germline transcripts (GLTs) were analyzed at 72 h postinfection with the results described above, Aicda and several IgH genes following sorting of infected cells based on mCherry expression. For quanti- were among the most severely decreased genes (Fig. 3A). In- tative PCRs, the pMIR sample was set to 1 (n =3).*p , 0.05, ***p , 0.001. terestingly, Batf and Irf4, two genes known to be involved in ns, not significant. regulating AID expression (27, 28), were also significantly The Journal of Immunology 2247 downregulated (Fig. 3A); this observation was independently We next assessed the effect of Sox2 deletion on AID levels. confirmed at the mRNA and protein levels (Fig. 3B, 3C). For these mechanistic studies, we deleted Sox2 from splenic Several genes involved in B cell activation and terminal dif- B cells derived from Sox2flox/flox mice via retroviral transduc- ferentiation were also differentially expressed, although no tion of the Cre-recombinase (Fig. 4A). Sox2flox/flox B cells in- clear pattern emerged to suggest differentiation into memory fected with retroviral Cre underwent a similar frequency of or plasma cells (Fig. 3A). Overall, the RNA-Seq analysis CSR to IgG1 as did uninfected cells (Fig. 4B), even though suggested that Sox2 could regulate multiple aspects of CSR, Sox2 expression was undetectable (Fig. 4C, 4D). However, including transcription of Aicda. there was a clear increase in AID protein and Aicda mRNA in Sox2-deleted B cells (Fig. 4D, 4E). Deletion of Sox2 increases IgH:c-Myc translocations To assess whether Sox2 deletion induces genomic instability, To determine whether Sox2 is expressed in B cells in vivo, mice we determined the frequency of IgH:c-Myc translocation, an were immunized with the model T-dependent Ag NP-CGG, established parameter of AID-dependent genome destabili- and GC B cells were purified (Supplemental Fig. 2A). Sox2 zation in B cells (18, 30). There was a significant increase in and AID were detectable in the GC B cell fraction but not in the frequency of IgH:c-Myc translocations upon loss of Sox2 the non-GC B cell population (Supplemental Fig. 2B). To (Fig. 4F, 4G). These results provide strong evidence that Sox2 assess the effect of Sox2 deletion during an immune response, represses AID expression once B cells have undergone CSR to Sox2flox mice, in which the coding sequence of Sox2 was promote genomic integrity.

flanked by loxP sites (14), were bred to AID-cre–transgenic Collectively, our data show that Sox2 is expressed in acti- Downloaded from mice, in which the Aicda transgene drives expression of the vated B cells and negatively regulates expression of AID fol- Cre-recombinase in activated B cells (29). GC B cells from lowing completion of CSR. The kinetics of Sox2 expression in Sox2flox/flox:AID-cre mice exhibited similar frequency of CSR B cells also suggests that it could be relevant in the setting of to IgG1 and NP-responsiveness relative to control mice reprogramming. Numerous studies demonstrated that AID is (Supplemental Fig. 2C). Additionally, serum Ig levels in involved in active demethylation processes in artificial settings, flox/flox Sox2 :AID-cre mice were indistinguishable from con- such as heterokaryon formation and transcription factor– http://www.jimmunol.org/ trols (Supplemental Fig. 2D). mediated reprogramming, as well as in the natural processes by guest on September 25, 2021

FIGURE 4. Loss of Sox2 increases IgH:c-Myc translocations. (A) Strategy to delete Sox2 in ex vivo–stimulated splenic B cells. B cells were harvested from wild- type (Sox2wt/wt)orSox2flox/flox (Sox2fl/fl) mice, activated with LPS+IL-4, and retrovirally infected with virus expressing Cre-recombinase and a GFP marker. (B) CSR to IgG1 was assayed by flow cytometry and quantified (n = 3). (C) Cells were harvested and analyzed by immunoblotting for expression of Sox2, AID, and tubulin (loading control). The asterisk (*) denotes a background polypeptide that sometimes reacted with Sox2 Abs. (D) Immunoblot with 2-fold dilution of protein extracts to assess AID levels. The four lanes for each genotype represent 10, 5, 2.5, and 1.25 mg of extract. (E) RNA from infected cells was analyzed for Aicda levels by quantitative PCR relative to b-actin; expression of wild-type B cells was normalized to 1. (F) Genomic DNA from activated splenic B cells was subjected to two rounds of PCR, resolved on agarose gel, stained with ethidium bromide (EtBr panel), and analyzed by Southern blot with IgH and c-Myc probes to assess translocation frequency. (G) Translocation frequency (n = 3). 2248 CUTTING EDGE: Sox2 REGULATES AID EXPRESSION occurring in fertilized oocytes and primordial germ cells (31– 15. Pucella, J. N., W. F. Yen, M. V. Kim, J. van der Veeken, C. T. Luo, N. D. Socci, Y. Naito, M. O. Li, N. Iwai, and J. Chaudhuri. 2015. miR-182 is largely dis- 34). AID also was implicated in the demethylation events that pensable for adaptive immunity: lack of correlation between expression and func- occur as part of the epithelial–mesenchymal transition during tion. [Published erratum appears in 2015 J. Immunol. 195: 1903.] J. Immunol. 194: 2635–2642. development and in tumorigenesis (35). For a potent mutator 16. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F. W. Alt. 2003. like AID to be involved in these natural processes, it is logical Transcription-targeted DNA deamination by the AID antibody diversification en- to think that the cell would want a system in place to prevent zyme. Nature 422: 726–730. 17. Nowak, U., A. J. Matthews, S. Zheng, and J. Chaudhuri. 2011. The splicing reg- unwanted AID activity once its role in demethylation is ulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of completed. Our recent data showing that AID expression is AID to switch-region DNA. Nat. Immunol. 12: 160–166. 18. Ramiro, A. R., M. Jankovic, T. Eisenreich, S. Difilippantonio, S. Chen-Kiang, only needed in the early phase of transcription factor–mediated M. Muramatsu, T. Honjo, A. Nussenzweig, and M. C. Nussenzweig. 2004. AID is reprogramming further suggests a need to limit its expression required for c-myc/IgH chromosome translocations in vivo. Cell 118: 431–438. 19. Chou, M. Y., F. W. Hu, C. H. Yu, and C. C. Yu. 2015. Sox2 expression in- (33). Therefore, the data presented in this article have impli- volvement in the oncogenicity and radiochemoresistance of oral cancer stem cells. cations for understanding termination of CSR, as well as in the Oral Oncol. 51: 31–39. field of reprogramming. 20. Chiles, T. C. 2004. Regulation and function of cyclin D2 in B lymphocyte subsets. J. Immunol. 173: 2901–2907. 21. Solvason, N., W. W. Wu, D. Parry, D. Mahony, E. W. Lam, J. Glassford, G. G. Klaus, P. Sicinski, R. Weinberg, Y. J. Liu, et al. 2000. Cyclin D2 is essential Acknowledgments for BCR-mediated proliferation and CD5 B cell development. Int. Immunol. 12: 2 2 We thank T. Honjo for AID / mice and members of the Chaudhuri 631–638. 22. Muramatsu, M., V. S. Sankaranand, S. Anant, M. Sugai, K. Kinoshita, laboratory for comments and discussion. N. O. Davidson, and T. Honjo. 1999. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in Downloaded from germinal center B cells. J. Biol. Chem. 274: 18470–18476. Disclosures 23. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, The authors have no financial conflicts of interest. M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al. 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102: 565–575. 24. Basso, K., and R. Dalla-Favera. 2010. BCL6: master regulator of the germinal References center reaction and key oncogene in B cell lymphomagenesis. Adv. Immunol. 105: 1. Hwang, J. K., F. W. Alt, and L. S. Yeap. 2015. Related mechanisms of antibody 193–210. http://www.jimmunol.org/ somatic hypermutation and class switch recombination. Microbiol. Spectr. 3: 325–348. 25. Medvedovic, J., A. Ebert, H. Tagoh, and M. Busslinger. 2011. Pax5: a master 2. Stavnezer, J., J. E. Guikema, and C. E. Schrader. 2008. Mechanism and regulation regulator of B cell development and leukemogenesis. Adv. Immunol. 111: 179–206. of class switch recombination. Annu. Rev. Immunol. 26: 261–292. 26. Singh, H., K. L. Medina, and J. M. Pongubala. 2005. Contingent gene regulatory 3. Nussenzweig, A., and M. C. Nussenzweig. 2010. Origin of chromosomal translo- networks and B cell fate specification. Proc. Natl. Acad. Sci. USA 102: 4949–4953. cations in lymphoid cancer. Cell 141: 27–38. 27. Fillatreau, S., and A. Radbruch. 2006. IRF4 - a factor for class switching and an- 4. Matthews, A. J., S. Zheng, L. J. DiMenna, and J. Chaudhuri. 2014. Regulation of tibody secretion. Nat. Immunol. 7: 704–706. immunoglobulin class-switch recombination: choreography of noncoding tran- 28. Ise, W., M. Kohyama, B. U. Schraml, T. Zhang, B. Schwer, U. Basu, F. W. Alt, scription, targeted DNA deamination, and long-range DNA repair. Adv. Immunol. J. Tang, E. M. Oltz, T. L. Murphy, and K. M. Murphy. 2011. The transcription 122: 1–57. factor BATF controls the global regulators of class-switch recombination in both 5. Campolo, F., M. Gori, R. Favaro, S. Nicolis, M. Pellegrini, F. Botti, P. Rossi, B cells and T cells. Nat. Immunol. 12: 536–543. E. A. Jannini, and S. Dolci. 2013. Essential role of Sox2 for the establishment and 29. Robbiani, D. F., A. Bothmer, E. Callen, B. Reina-San-Martin, Y. Dorsett, maintenance of the germ cell line. Stem Cells 31: 1408–1421. S. Difilippantonio, D. J. Bolland, H. T. Chen, A. E. Corcoran, A. Nussenzweig, and by guest on September 25, 2021 6. Kamachi, Y., M. Uchikawa, A. Tanouchi, R. Sekido, and H. Kondoh. 2001. Pax6 M. C. Nussenzweig. 2008. AID is required for the chromosomal breaks in c-myc and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens that lead to c-myc/IgH translocations. Cell 135: 1028–1038. development. Genes Dev. 15: 1272–1286. 30. Ramiro, A. R., M. Jankovic, E. Callen, S. Difilippantonio, H. T. Chen, 7. Que, J., T. Okubo, J. R. Goldenring, K. T. Nam, R. Kurotani, E. E. Morrisey, K. M. McBride, T. R. Eisenreich, J. Chen, R. A. Dickins, S. W. Lowe, et al. 2006. O. Taranova, L. H. Pevny, and B. L. Hogan. 2007. Multiple dose-dependent roles Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. for Sox2 in the patterning and differentiation of anterior foregut endoderm. De- Nature 440: 105–109. velopment 134: 2521–2531. 31. Bhutani, N., J. J. Brady, M. Damian, A. Sacco, S. Y. Corbel, and H. M. Blau. 2010. 8. Lesko, M. H., R. R. Driskell, K. Kretzschmar, S. J. Goldie, and F. M. Watt. 2013. Reprogramming towards pluripotency requires AID-dependent DNA demethyla- Sox2 modulates the function of two distinct cell lineages in mouse skin. Dev. Biol. tion. Nature 463: 1042–1047. 382: 15–26. 32. Bombardieri, M., F. Barone, F. Humby, S. Kelly, M. McGurk, P. Morgan, 9. Pevny, L. H., and S. K. Nicolis. 2010. Sox2 roles in neural stem cells. Int. J. Bio- S. Challacombe, S. De Vita, G. Valesini, J. Spencer, and C. Pitzalis. 2007. chem. Cell Biol. 42: 421–424. Activation-induced cytidine deaminase expression in follicular dendritic cell net- 10. Xia, P., S. Wang, B. Ye, Y. Du, G. Huang, P. Zhu, and Z. Fan. 2015. Sox2 works and interfollicular large B cells supports functionality of ectopic lymphoid functions as a sequence-specific DNA sensor in neutrophils to initiate innate im- neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjo¨gren’s syn- munity against microbial infection. Nat. Immunol. 16: 366–375. drome. J. Immunol. 179: 4929–4938. 11. Yamanaka, S., and H. M. Blau. 2010. Nuclear reprogramming to a pluripotent state 33. Kumar, R., L. DiMenna, N. Schrode, T. C. Liu, P. Franck, S. Mun˜oz-Descalzo, by three approaches. Nature 465: 704–712. A. K. Hadjantonakis, A. A. Zarrin, J. Chaudhuri, O. Elemento, and T. Evans. 2013. 12. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and T. Honjo. AID stabilizes stem-cell phenotype by removing epigenetic memory of pluripotency 2000. Class switch recombination and hypermutation require activation-induced genes. Nature 500: 89–92. cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553–563. 34. Popp, C., W. Dean, S. Feng, S. J. Cokus, S. Andrews, M. Pellegrini, S. E. Jacobsen, 13. Vuong, B. Q., M. Lee, S. Kabir, C. Irimia, S. Macchiarulo, G. S. McKnight, and and W. Reik. 2010. Genome-wide erasure of DNA methylation in mouse pri- J. Chaudhuri. 2009. Specific recruitment of protein kinase A to the immunoglob- mordial germ cells is affected by AID deficiency. Nature 463: 1101–1105. ulin locus regulates class-switch recombination. Nat. Immunol. 10: 420–426. 35. Mun˜oz, D. P., E. L. Lee, S. Takayama, J. P. Coppe´, S. J. Heo, D. Boffelli, J. M. Di 14. Shaham, O., A. N. Smith, M. L. Robinson, M. M. Taketo, R. A. Lang, and Noia, and D. I. Martin. 2013. Activation-induced cytidine deaminase (AID) is R. Ashery-Padan. 2009. Pax6 is essential for lens fiber cell differentiation. Devel- necessary for the epithelial-mesenchymal transition in mammary epithelial cells. opment 136: 2567–2578. Proc. Natl. Acad. Sci. USA 110: E2977–E2986.