Activation-induced cytidine deaminase (AID)- dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1 Yuichi Kanehiroa,1,2, Kagefumi Todoa,1,3, Misaki Negishia, Junji Fukuokaa, Wenjian Ganb, Takuya Hikasaa, Yoshiaki Kagaa, Masayuki Takemotoa, Masaki Magaria, Xialu Lib, James L. Manleyc,4, Hitoshi Ohmoria,4, and Naoki Kanayamaa,4 aDepartment of Bioscience and Biotechnology, Okayama University Graduate School of Natural Science and Technology, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan; bNational Institute of Biological Sciences, Beijing 102206, People’s Republic of China; and cDepartment of Biological Science, Columbia University, New York, NY 10027 Contributed by James L. Manley, December 12, 2011 (sent for review October 9, 2011) Somatic hypermutation (SHM) of Ig variable region (IgV) genes DNAs harboring SHM hotspot motifs, thus enabling AID-me- requires both IgV transcription and the enzyme activation-induced diated deamination (8, 23–25). In addition, it has recently been cytidine deaminase (AID). Identification of a cofactor responsible reported that depletion of the THO-TREX complex, which for the fact that IgV genes are much more sensitive to AID-induced functions as the interface between transcription and mRNA ex- mutagenesis than other genes is a key question in immunology. port, enhances mutation in AID-expressing yeast cells (26). Here, we describe an essential role for a splice isoform of the pro- However, cofactors that are essential for generating and stabi- lizing ssDNA bubbles in the IgV genes in vivo are not well de- totypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, fi in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, ned, although these recent reports suggest that transcription- coupled factors have a role in generating AID substrates. we found that SHM does not occur in a DT40 line lacking SRSF1-3 Chicken DT40 B cells provide an excellent system to analyze (DT40-ASF), although it is readily detectable in parental DT40 cells. AID cofactors responsible for SHM. These cells spontaneously Strikingly, overexpression of AID in DT40-ASF cells led to a large fi undergo Ig hypermutation by AID-dependent gene conversion IMMUNOLOGY increase in nonspeci c (off-target) mutations. In contrast, intro- (GCV) and point mutation (27–30), the latter being equivalent fi duction of SRSF1-3, but not SRSF1, into these cells speci cally re- to SHM observed in human and murine B cells (31). In- stored SHM without increasing off-target mutations. Furthermore, terestingly, it has been shown that genetic depletion of a serine/ we found that SRSF1-3 binds preferentially to the IgV gene and arginine (SR)-rich protein splicing factor SRSF1 in a DT40 cell inhibits processing of the Ig transcript, providing a mechanism by line (DT40-ASF) results in enhanced genomic instability because which SRSF1-3 makes the IgV gene available for AID-dependent of generation of ssDNA in transcribed regions genome-wide SHM. SRSF1 not only acts as an essential splicing factor but also (32). A prototypical SR protein, SRSF1 [formerly ASF/SF2 regulates diverse aspects of mRNA metabolism and maintains ge- (33)], not only acts as an essential splicing factor but also exerts nome stability. Our findings, thus, define an unexpected and impor- biological roles in diverse aspects of RNA metabolism, including tant role for SRSF1, particularly for its splice variant, in enabling AID mRNA nuclear export, mRNA stability, mRNA quality control, to function specifically on its natural substrate during SHM. and regulation of translation (34, 35). In this study, we used DT40 cell lines to analyze requirements fi fi gene conversion | genomic instability for accurate and ef cient AID-dependent SHM. Signi cantly, we found that SRSF1, and in particular a specificspliceisoform, SRSF1-3, is necessary for the AID-dependent SHM machinery to omatic hypermutation (SHM) and class switch recombination target the IgV genes. Our results, thus, not only identify a cofactor S(CSR) of Ig genes are key processes needed to generate fi necessary for ensuring accurate SHM but also expand the role of functional antibodies (Abs) with high af nity in humoral immune SR proteins to include a function in the immune response. responses. Activation-induced cytidine deaminase (AID) is cru- cial for both SHM and CSR (1, 2), which are initiated by AID- Results catalyzed deamination of dC to dU in the variable (V) region and Hypermutation Is Deficient in DT40-ASF Cells. In DT40-ASF cells, the switch (S) region DNA strands, respectively (3, 4). However, the endogenous SRSF1 gene is homozygously disrupted, and AID has been recently shown to bind numerous chromosomal a human SRSF1 cDNA under the control of a tetracycline (Tet)- locations genome-wide and to mutate non-Ig genes, although the repressible promoter is present as the only source of SRSF1 (36). mutation frequency of non-Ig genes is much lower than that of the When DT40-ASF cells are treated with Tet or its more active fi Ig genes (5, 6). It has been proposed that speci c cofactors interact analog, doxycycline (Dox), most of cells die because of depletion with AID and regulate its activity in a target-specificmanner(7). AID has been reported to associate with several proteins including RPA (8, 9), protein kinase A (10), RNA polymerase II (Pol II) – Author contributions: H.O. and N.K. designed research; Y. Kanehiro, K.T., M.N., J.F., W.G., (11), DNA-PKcs (12), MDM2 (13), Spt6 (14), 14 3-3 (15), Spt5 T.H., Y. Kaga, M.T., M.M., and N.K. performed research; X.L. and J.L.M. contributed new (16), CTNNBL1 (17), PTBP2 (18), RNA exosome subunits (19), reagents/analytic tools; Y. Kanehiro, K.T., X.L., J.L.M., H.O., and N.K. analyzed data; and KAP1 (20), and HP1 (20). However, these investigations were J.L.M., H.O., and N.K. wrote the paper. mostly focused on CSR, and, thus, the identified factors them- The authors declare no conflict of interest. selves are not sufficient to explain how AID activity is specifically 1Y.K. and K.T. contributed equally to this work. targeted to the IgV genes during SHM. 2Present address: Department of Microbiology and Immunology, Shimane University SHM occurs in IgV region exons starting 100–200 bp down- School of Medicine, Izumo, Shimane 693-8501, Japan. – stream of the promoter and extending over 1 2 kb, preferentially 3Present address: Center for Innovation in Immunoregulative Technology and Therapeu- targeting WRC/GYW hotspot motifs (3, 4). Importantly, the tics, Kyoto University Graduate School of Medicine, Sakyo-Ku, Kyoto 606-8501, Japan. activity of the SHM machinery strictly depends on transcription 4To whom correspondence may be addressed. E-mail: [email protected], hit2224@cc. of the IgV genes (21, 22). In vitro experiments have shown that okayama-u.ac.jp, or [email protected]. replication protein A (RPA) binds to and stabilizes single- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stranded (ss)DNA bubbles in transcribed artificial substrate 1073/pnas.1120368109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1120368109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 of SRSF1, an essential factor in diverse aspects of RNA me- An SRSF1 Splice Isoform, SRSF1-3, but Not SRSF1, Is Required for tabolism. However, a small fraction of cells proliferate as Tet (or Hypermutation in the IgV Genes. We next sought to investigate the Dox)-resistant colonies, in which SRSF1 expression escaped possible basis for the defect in SHM in DT40-ASF cells. One from the control of the Tet-Off promoter (32). This has been possibility may reflect the fact that these cells possess only hu- shown to reflect enhanced DNA rearrangement and trans- man SRSF1 instead of the chicken ortholog (Fig. 1B) (36). location of the exogenous SRSF1 gene, resulting from the for- However, hypermutation potency was not restored when the mation of cotranscriptional R loops under SRSF1-deficient chicken SRSF1 was reintroduced in DT40-ASF at a comparable conditions. To explore a role for SRSF1 in the formation of level to that observed in the wild type (Fig. 2 A and B and Table transcription bubbles during SHM, we first analyzed a hyper- S1). The chicken SRSF1 locus, as shown in human and mouse mutation profile of the IgV gene in DT40-ASF cells that has been cell lines, can generate not only full-length SRSF1 but also splice maintained in the absence of Tet and Dox. Unexpectedly, we variants including SRSF1-2 and SRSF1-3 (formerly ASF-2 and found that the DT40-ASF cell population bore a virtually ASF-3, respectively), the functions of which remain unknown monoclonal IgV gene (Fig. S1A), indicating that DT40-ASF has (37, 38) (Fig. 1C). We confirmed that DT40-ASF cells did not a critical defect in GCV and SHM. To further confirm the defect express the chicken isoforms of these transcripts (Fig. 1D), fi of IgV diversi cation in DT40-ASF cells, hypermutation fre- whereas wild-type DT40 cells, as well as chicken and mouse quencies in the IgVL gene were compared between wild-type lymphoid tissues, expressed SRSF1 and SRSF1-3 transcripts (Fig. DT40 and DT40-ASF cells after culture for 30 d. Strikingly, 1 D and E and Fig. S2). Thus, we considered the possibility that DT40-ASF cells were unable to introduce mutations (Fig. 1A and Fig. S1B), despite comparable expression of AID and SRSF1, relative to the mutation-competent wild type (Fig. 1B). 1.2 4.0 A SRSF1 SRSF1-3 3.0 A DT40-WT DT40-ASF 0.8 3 5 2.0 Relative 2 Relative 0.4 Non-mutated 1.0