Activation-induced cytidine deaminase (AID)- dependent 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 . 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 (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 1.0 154 175 21 85 transcription level Mutated transcription level 0 0.0 1 WT #1 #2 Vector WT #1 #2 Vector 0 0 SRSF1R SRSF1-3R Mutation events DT40-ASF DT40-ASF /bp (x10-4) 4.3 0 10.0 2.0 1.5 WT DT40-ASF B R R B AID SRSF1 8.0 SRSF1 SRSF1-3 AID 1 2 3 1.5 ) 6.0 1

1.0 -4 β-actin 1.0 4.0 (x10

Relative 0.5 0.5 HA-hSRSF1 2.0 109 125

cSRSF1 Mutation events/bp transcription level 0.0 0.0 0.0 R R WT DT40 WT DT40 β-actin 0 0 -ASF -ASF Vector SRSF1 SRSF1-3 C SRSF1 locus D DT40-WT DT40-ASF exon1 exon2 exon3 exon4 C #1 #2 #3 SRSF1-3 a/b CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 SRSF1 0 300 0 300 0 300 c b a/c transcription SRSF1-3 ST β-actin SRSF1 ST 5.0 9/55 7/48 10/48 SRSF1-2 E SRSF1 ST Mutants/Analyzed clones SRSF1-3 SRSF1-3 4.0 (preSRSF1) D translation 3.0 2.0 DT40-ASF RRM1 RRM2 RS AID SRSF1 2.0 1.5 SRSF1-3R Relative SRSF1-2 AID-KOVector SRSF1R #1 #2 1.0 1.0 SRSF1-2C-terminus transcription level AID

SRSF1-3 0.0 Relative 0.5 SRSF1-3C-terminus WTASF SP BM BF TH β-actin DT40 transcription level 0.0 R #1 #2 N/A 1.0 4.1 2.2 2.5 Fig. 1. DT40-ASF cells lack IgV hypermutation activity even in the presence Vector SRSF1-3R AID-KO SRSF1 of AID expression. (A) IgVL sequence analysis of wild-type DT40 and DT40- DT40-ASF ASF cells after culture for 30 d. The numbers of mutated and nonmutated genes are indicated in each pie chart. Segment sizes in the satellite chart Fig. 2. The SRSF1 splice isoform SRSF1-3 restores hypermutation in DT40- depict the proportion of sequences containing 1–5 mutations. (B) Quanti- ASF cells. (A) Levels of chicken SRSF1 and SRSF1-3 transcripts in DT40-ASF tative RT-PCR (Left) and Western blot (Right) analyses of AID and SRSF1 cells transfected with a mock vector or the chicken SRSF1 or SRSF1-3 cDNA expression. Primers that can bind to both chicken and human SRSF1 genes (Vector, SRSF-1R, or SRSF1-3R, respectively). SRSF1 and SRSF1-3 transcript were used. Data are means of triplicates; error bar, SD. (C) The structure of levels were estimated by quantitative PCR using primers specific for chicken the chicken SRSF1 locus (NC_006106.2) and its products. Splice variants are SRSF1 and SRSF1-3. Data were normalized to levels of the β-actin transcript. generated from the chicken SRSF1. RRM, RNA-recognition motif; RS, argi- Two independent clones were analyzed. (B) IgVL sequence analysis of nine/serine-rich region; S, start; T, termination. (D) RT-PCR of chicken SRSF1 transfected DT40-ASF cells. The mutation frequency is the mean ± SD (n =2 and SRSF1-3 transcripts. Fourfold serially diluted cDNAs were used. Primers or 3). Pie charts are displayed as in Fig. 1A.(C) Mutated sequences are il- are shown as arrowheads in A.(E) Expression of chicken SRSF1 and SRSF1-3 lustrated as horizontal bars. Sticks and balls, point mutations; bold bars, GCV transcripts in the bone marrow (BM), bursa of Fabricius (BF), spleen (SP), and tracts. The number of independent mutants in total analyzed clones is thymus (TH) cells removed from a chicken and in the wild-type DT40 and shown below. (D) Levels of the AID transcript and protein were assessed with DT40-ASF cells. Data were collected using quantitative RT-PCR and normal- quantitative RT-PCR and Western blotting, respectively. β-actin was used as ized to the levels of the β-actin and gapdh transcripts. a control. Relative protein levels to Vector are indicated below.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1120368109 Kanehiro et al. Downloaded by guest on September 25, 2021 a splice variant other than SRSF1 is necessary for hypermutation A IgL locus B 2.0 in DT40 cells. Pseudo genes SRSF1 fi SRSF1-3 To test the above idea, we rst transfected DT40-ASF cells r 1.5 with a chicken splice variant cDNA, preSRSF1. The preSRSF1 C GFP BFP BSR cDNA can produce SRSF1 and SRSF1-2 after splicing and also GFP BFP 1.0 199 199 directly generate SRSF1-3 without splicing (Fig. 1C). This led to Relative fi TA C T 0.5 a signi cant restoration of both GCV and SHM (Fig. S3). Im- Blue emission transcription level portantly, when DT40-ASF cells were transfected with a CMV 199T 0.0 #1 #2 #1 #2 promoter-driven SRSF1-3 encoding cDNA, the frequency of TA T T WT SRSF1-3O/E both GCV and SHM in the IgVL gene was completely restored Green emission to the level observed in wild-type cells (Fig. 2 and Table S1). 7 However, an SRSF1-3 cDNA mutant in which the ATG start C WT SRSF1-3O/E D 6 codon was changed to CTG failed to repair the defect of IgV 0% 0% hypermutation in DT40-ASF. Overexpression of SRSF1-3 did AID-OFF 5 not increase the expression level of AID (Fig. 2D), and resto- 4 cells (%) ration of hypermutation activity was also observed in the IgVH 3 high

gene in the SRSF1-3-reconstituted DT40-ASF cells (Fig. S4). FSC 1.6% 0.2% 2

We next sought to determine whether increased expression of GFP SRSF1-3 also enhances hypermutation in wild-type DT40 cells AID-ON 1 and whether the enhancement of hypermutation depends on 0 AID - + -+ + -+ - AID. To this end, we examined the effect of overexpression of GFP #1 #2 #1 #2 SRSF1-3 on GCV frequency in an engineered line, DT40-SW, WT SRSF1-3O/E which harbors an artificial GCV substrate (39) and the intact SRSF1 locus and in which AID expression can be switched on E 10.0 3 and off (40). In this cell line, a GCV event in each cell can be fl 7.5 monitored as the change of uorescence color from blue to 2 green (Fig. 3A). When the GCV substrate, G/B construct, was placed in the IgVL locus in DT40-SW cells, overexpression of 5.0 cells (%) high

SRSF1-3 led to enhancement of GCV frequency in an AID- 1 IMMUNOLOGY 2.5 dependent fashion (Fig. 3 B–D). Transfection with the SRSF1-2 GFP cDNA was, however, ineffective. Taken together, we conclude Relative transcription level 0.0 0 that the SRSF1-3 protein has a critical role in inducing AID- #1 #2 #1 #2 WT dependent hypermutation in the IgV genes. SRSF1-3O/E G/B OVA OVA IgL WT SRSF1-3O/E AID Functions in SRSF1 Depletion-Induced Genomic Instability. In Fig. 3. SRSF1-3 enhances AID-dependent hypermutation on the IgV locus in addition to diversifying the Ig genes, AID can also target a vari- fi ety of non-Ig genes (5), in some cases causing malignant trans- DT40 cells bearing the intact SRSF1 locus. (A) An arti cial GCV substrate, G/B construct. G/B construct was introduced in the IgL locus in DT40-SW cells. formation by initiating chromosome translocations (41). By Conversion of 199C to T in the transcribed EBFP gene by GCV with the up- treating DT40-ASF cells with Tet or Dox, DNA rearrangement stream untranscribed EGFP gene results in fluorescence shift from blue to and translocation of the exogenous SRSF1 gene can be induced green. Expression of the EBFP gene is driven by the CMV promoter and under SRSF1-deficient conditions, resulting from the formation a synthetic intron. (B) The levels of chicken SRSF1 and SRSF1-3 transcripts in of cotranscriptional R loops (32). Although we have recently DT40-SW cells transfected with the SRSF1-3 cDNA were assessed by quanti- shown that this reflects, in part, defects in DNA replication (42), tative RT-PCR. Two independent transfectants (indicated as SRSF1-3O/E) were we next examined whether AID also contributes to SRSF1 de- analyzed. Data were normalized to the level of the β-actin transcript. (C) pletion-induced genomic instability, which can be observed as Flow-cytometric analysis of GCV frequency. AID-expressing (AID-ON) cells occurrence of Tet- or Dox-resistant colonies. When AID levels were cultured for 30 d and analyzed for occurrence of GCV (GFPhigh cells). (D) were reduced by stable expression of AID-specific shRNAs, the SRSF1-3 overexpression enhanced mutation frequency in the IgVL locus. appearance of Tet-resistant clones following SRSF1 depletion Single-cell-sorted subclones of DT40-SW cells bearing the G/B construct in was suppressed (Fig. 4A). On the other hand, in DT40-ASF the IgVL locus (AID-OFF cells, n = 2; AID-ON cells, n =21to∼32) were ana- cells overexpressing AID, the frequency of Dox-resistant colony lyzed. (E) SRSF1-3 overexpression did not induce hypermutation in a non-Ig formation sharply increased (Fig. 4B), possibly because of an locus. Wild-type DT40 cells in which the G/B construct was introduced in the increased attack of AID to non-Ig targets (ssDNA regions or ovalbumin locus were transfected with the SRSF1-3 cDNA and single cells fi (n = 24) were analyzed after culture for 3 wk (Right). The level of the SRSF1-3 R-loops) generated under SRSF1-de cient conditions. The re- transcript in transfectants was determined by quantitative RT-PCR (Left). sultant colonies expressed SRSF1 even in the presence of Dox. The apparent lower frequency of colony formation in the pres- ence of Dox (Fig. 4 B and C, Vector) may reflect the fact that in the original or AID-overexpressing cells under SRSF1-de- Dox is ∼100-times more effective than Tet (43) and more readily pleted conditions (Fig. 4C). If overexpression of SRSF1-3 causes causes cell death at the concentrations used. In any event, these genomic instability in the presence of SRSF1, drug-resistant cells results are consistent with previous findings that AID is involved in should accumulate spontaneously during maintenance in culture translocation of the c-myc gene to the IgH locus in B without addition of Dox. However, this was not the case, as (41) and that AID expression leads to induction of genome-wide observed in SRSF1-3-reconstituted DT40-ASF cells in compar- mutations (5). Thus, our data suggest that SRSF1 naturally pro- ison with the vector control (Fig. 4C). In addition, reporter- tects against the off-target action of AID, likely by preventing based assays for GCV using the G/B construct showed that appearance of ssDNA as a result of R-loop formation. overexpression of SRSF1-3 in wild-type DT40 cells did not in- duce GCV in the reporter construct placed in the ovalbumin SRSF1-3 Has a Critical Role in Targeted Induction of Hypermutation in locus, in which the AID-dependent hypermutation machinery the IgV Genes. We next investigated whether SRSF1-3 contributes has been shown to be inactive (39) (Fig. 3E). These results in- to genetic alteration of non-Ig targets. We used an SRSF1-depletion dicate that SRSF1-3 did not contribute to off-target mutation assay in DT40-ASF cells overexpressing AID and/or SRSF1-3. In induced by AID either in the presence or absence of SRSF1. contrast to overexpression of AID, enhanced SRSF1-3 expres- On the other hand, when DT40-ASF cells were treated with sion did not further increase the translocation frequency either Dox for 48 h followed by culture in the absence of Dox for 1 mo,

Kanehiro et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 mutations were not found in the IgV genes of these cells, al- though the Dox treatment has been shown sufficient to cause A DT40 AID KD 100 -ASF B43 B53 Cells/well genomic instability (32). In addition, despite the enormous in- AID 75 20 crease of translocation, AID-overexpressing DT40-ASF cells β-actin 200 showed only a marginal increase in IgVL mutations, most of RT-PCR 50 2000 which were limited to point mutations (Fig. 4D and Fig. S5). DT40 AID KD However, introduction of SRSF1-3 into these cells resulted in -ASF B43 B53 25 β-actin restoration of the hypermutation frequency to the level of the 0 0 00 00 wild-type cells, in terms of both GCV and SHM (Fig. 4D and Fig. AID

Tet-resistant colonies (%) Tet-resistant S5). Taken together, our data suggest that SRSF1-3 has a role in Western blot DT40 B43 B53 -ASF AID KD the specific induction of AID activity in the IgV genes. B 40 SRSF1-3 Collaborates with AID to Inhibit Splicing of Ig Transcripts. AID How does SRSF1-3 direct AID to act on the IgV gene? To ad- 30 100 Cells/well dress this issue, we examined whether AID and SRSF1-3 are 20 20 75 recruited to the IgV gene. To discriminate between SRSF1 and 10 Relative 200 SRSF1-3 in DT40 cells, we introduced Flag-tagged SRSF1-3 into 0 50 2000 DT40-ASF cells. Chromatin immunoprecipitation (ChIP) anal- WT DT40 #1 #2 transcription level -ASF AIDO/E 20000 ysis using an anti-Flag mAb revealed that SRSF1-3 localized O/E 25 weakly but preferentially to the IgV gene (Fig. 5 A and C; the DT40 AID fl -ASF #1 #2 00 weak signal likely re ects a suppressed level of SRSF1-3 protein 0 in the presence of SRSF1; Fig. S6). With respect to AID re- AID colonies (%) Dox-resistant Vector #1 #2 β-actin AIDO/E cruitment to the IgV gene, in vivo evidence has been limited Western blot except for experiments using Ramos human cells C (44). By careful optimization of ChIP conditions, a weak AID signal was detected on the IgV gene, and this was not further 100 Cells/well enhanced by SRSF1-3 expression (Fig. 5 B and C). These results 75 20 suggest that SRSF1-3 does not participate in AID recruitment to 200 the IgV genes. 50 2000 In vitro biochemical analyses have shown that SRSF1-3 not only lacks splicing activity but also inhibits the activity of SRSF1 25 (45). Thus, SRSF1-3 might function as a competitive inhibitor of

resistant colonies (%) 00 00 SRSF1 in vivo. Indeed, introduction of SRSF1-3 resulted in an 0 increased nuclear accumulation of unspliced nascent Ig tran-

Dox- Vector #1 #2 Vector #1 #2 SRSF1-3R SRSF1-3R scripts in DT40-ASF cells (Fig. 6A). Interestingly, the unspliced AIDO/E RNA accumulation was largely dependent on AID expression D (Fig. 6B), implying a close relationship between unspliced Ig 12.0 RNA generation and the presence of AID. Below, we discuss AIDO/E O/E how accumulation of unspliced nascent Ig RNA facilitates AID- AID +SRSF1-3R ) 8.0 3 dependent SHM.

-4 2 2 1 1 Discussion (x10 4.0 AID-dependent SHM is largely limited to the IgV genes, al- 108 128 though off-target mutations and translocations that depend on Mutation events/bp 0.0 R AID are also found at much lower frequency (5, 6). What directs O/E O/E 0 0 Vector the SHM machinery to its targets is an important issue for un- AID AID derstanding not only SHM but also the mechanisms of AID- +SRSF1-3 linked oncogenic genomic alterations. However, reliable culture Fig. 4. SRSF1-3 does not contribute to an off-target activity of AID but conditions have not been established that enable naïve human or induces hypermutation specifically in the IgV gene. (A) AID is important for murine B cells to undergo SHM ex vivo. Thus, DT40 cells, which SRSF1 depletion-induced genomic instability. Two AID knockdown deriva- spontaneously undergo hypermutation, are useful for analyzing tives of DT40-ASF, B43 and B53, were established by stable transfection of mechanisms of SHM, because the Ig diversification process in shRNA targeting the AID gene. Steady-state levels of AID mRNA and protein chicken and mammals is similar (31). The present results have were determined by RT-PCR and Western blot (Left). SRSF1-depeletion assay established an essential role for a splicing isoform of the SR was carried out using DT40-ASF and AID knock-down cells (Right). (B) protein SRSF1, SRSF1-3, in AID-induced SHM in the IgV genes Overexpression of AID enhances genomic instability. AID was overexpressed in DT40 cells. The specific action and localization of SRSF1-3 to in DT40-ASF cells by stable transfection of AID cDNA. Steady-state levels of the IgV genes leads us to the conclusion that SRSF1-3 plays AID mRNA and protein were determined by quantitative RT-PCR and a critical role in the targeted induction of AID activity specifi- Western blot (Left) (5- and 10-fold diluted samples were also loaded). SRSF1- cally in the IgV genes, probably by providing AID with substrate depeletion assay was carried out using AID-overexpressing DT40-ASF cells O/E ssDNA on the IgV genes by reducing Ig RNA splicing. (AID )(Right). (C) Overexpression of SRSF1-3 does not affect AID-induced Several AID cofactors have been reported to be essential for genomic instability. DT40-ASF cells overexpressing AID and/or SRSF1-3 (AIDO/E, R OE R AID-dependent SHM, GCV and CSR. However, there is little SRSF1-3 ,orAID +SRSF1-3 , respectively) were used for SRSF1-depeletion evidence for cofactors that restrict the activity of AID specifically assays. Two representative transfectants were assessed. DT40-ASF and AIDOE OE to the Ig genes, although Liu and Schatz have proposed that non- cells transfected with a mock vector (Vector and AID +Vector, respectively) fi were used as controls. Cells were treated with 1 μg/mL Tet (A) or 50 ng/mL Ig loci are protected from AID-induced SHM by high- delity DNA repair (46). The present study showed that constitutive Dox (B and C). The number 0 in the charts indicates that no Tet or Dox-re- fi sistant colonies formed. (D) SRSF1-3 but not AID induces hypermutation expression of AID was not suf cient for SHM induction in efficiently in the IgV gene in DT40-ASF cells. Two or three independent DT40-ASF cells, although the amount of AID was enough to clones of DT40-ASF cells overexpressing AID and/or SRSF1-3 (Vector, AIDOE, cause translocations when SRSF1 was depleted. Thus, in the and AIDOE+SRSF1-3R, respectively) were cultured for 30 d and IgVL sequences absence of SRSF1-3, the IgV genes behave similarly to non-Ig were analyzed. loci and are not subject to SHM. Because SRSF1-3 appeared not

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1120368109 Kanehiro et al. Downloaded by guest on September 25, 2021 5 A IgVH Histone H2A A Vector SRSF1-3R Vector F-SRSF1-3R Vector F-SRSF1-3R IgH locus RT– RT– 4 - actin

mAb L V D J C β L-V/J-C 3 Flag J-C DT40-ASF ab 2 Vector F-SRSF1-3R IgG β-actin 1

Input Transcripts/ 0 Vec SRSF Flag-SRSF1-3 GAPDH β-ACTIN 1-3R (25kD) Vector F-SRSF1-3R Vector F-SRSF1-3R 5 mAb B WT SRSF1-3O/E Flag 4 β AID – + – + - actin -actin β RT– RT– RT– RT– 3 IgG L-V/J-C J-C 2 Input β-actin 1 Transcripts/ 0 B IgVH Histone H2A AID – + – + DT40-ASF DT40-ASF WT SRSF 1-3O/E AID-KO Vector F-SRSF1-3R AID-KO Vector F-SRSF1-3R mAb Fig. 6. SRSF1-3 inhibits splicing of the Ig transcripts in an AID-dependent AID manner. (A) SRSF1-3 expression increased unspliced IgH RNAs in the nucleus. H4Ac Nuclear RNAs extracted from SRSF1-3R cells were used for analysis. IgH IgG transcripts with unspliced L-V and J-C introns or only J-C intron (designated as L-V/J-C and J-C, respectively) were analyzed by RT-PCR (Left). Positions of Input a sense primer for L exon (indicated as a) and antisense primers for J-C intron (indicated as b) are shown as arrowheads. Fourfold serially diluted cDNAs 8 20 5 were used for PCR. RNAs without reverse transcription were used as controls C IgVH IgVL IgVH ) IgG IgG IgG fi

-3 (RT-). The level of unspliced transcript was also quanti ed by quantitative 16 4 α 6 α Flag α Flag AID PCR (Right). (B) The increase of unspliced Ig transcripts depends on AID. RT- 12 3 PCR of unspliced Ig transcripts was carried out using SRSF1-3-overexpressing 4 DT40-SW cells (SRSF1-3O/E) with or without AID expression (Left). The level of IMMUNOLOGY 8 2 unspliced transcript was also quantified by quantitative PCR (Right). Data are 2 4 1 representative of two or more experiments. % of input (x10 0 0 0 R R R Vec F-SRSF1-3 Vec F-SRSF1-3 AID-KO Vec F-SRSF1-3 DT40-ASF DT40-ASF DT40-ASF formation that can be accessible by AID (26). In addition, it has Fig. 5. SRSF1-3 is localized on the IgV genes but does not enhance re- been recently reported that Spt5, a factor associated with stalled cruitment of AID to the IgV gene. (A) Expression of Flag-tagged SRSF1-3 in Pol II and ssDNA, is required for recruiting AID to target sites DT40-ASF. Flag-SRSF1-3 was detected by Western blot using anti-Flag anti- during CSR (16). During immune responses, centroblasts, a body. ChIP analysis for SRSF1-3. Crosslinked chromatin was extracted from major B-cell population undergoing SHM in germinal centers, R DT40-ASF cells expressing Flag-SRSF1-3 (F-SRSF1-3 ), precipitated using anti- are surface Ig-dim or negative (47). This in vivo observation is in Flag (clone M2), and examined for the IgVH and control genes by good agreement with recent reports and our findings in the PCR. Fourfold serially diluted ChIP DNAs were used for PCR. (B)ChIPanalysis R context of regulation of transcription and posttranscriptional for AID. Chromatin obtained from SRSF1-3 cells using anti-AID or anti-acet- processes of the Ig genes. ylated H4 antibodies was analyzed for the IgVH and H2A genes. Data are Hypermutation in DT40 cells has been estimated to occur representative of two or more experiments. (C) Quantitative PCR analysis of ChIP DNA for Flag-tagged SRSF1-3 and AID. Quantitative PCR was carried out approximately every 40 cell cycles (27). That is probably attrib- for the IgVH and IgVL genes for chromatin precipitated using an anti-Flag utable to a low level of SRSF1-3 protein. SRSF1 has been shown antibody (clone 1E6) and for the IgVH gene for chromatin obtained using an to autoregulate its own expression (48), and, thus, a relatively anti-AID antibody. Data were normalized for input DNA. Means ± SD of large amount of SRSF1 will suppress expression of SRSF1-3, triplicate assays are displayed. Data are representative of two experiments. consistent with our finding that the level of SRSF1-3 was dras- tically increased when SRSF1 was depleted. As a result, the level of unspliced Ig RNA in the nuclei of DT40 cells appeared to be to enhance recruitment of AID to the IgV genes, the SRSF1-3- very low. In good agreement with this, overexpression of SRSF1- induced activation of SHM specifically on the IgV genes must 3 in wild-type DT40 cells enhanced GCV frequency and gener- result from another activity of SRSF1-3, and we suggest that this ation of unspliced Ig RNA in an AID-dependent manner. Thus, reflects its inhibitory effect on SRSF1 (45). regulation of SRSF1-3 expression to low levels might result in We suggest that the enhanced accumulation of unspliced Ig stochastic occurrence of hypermutation in DT40 cells. transcripts by SRSF1-3 results from inhibition of SRSF1 activity Alternative splicing enables particular genes to produce two or by SRSF1-3. Depletion of SRSF1 results in the generation of more mRNAs, each of which can have different physiological cotranscriptional R loops, likely, at least in part, from an increase functions. SRSF1 was originally described as an alternative in unspliced nascent transcripts, suggesting that SRSF1 sup- splicing factor, but more recent studies have elucidated its roles presses transcription-linked ssDNA formation to maintain ge- in many aspects of RNA metabolism (34, 35). The present study nomic stability (32). Thus, an SRSF1-3-induced increase of not only reveals a function of SRSF1 in AID-dependent hyper- unspliced transcripts likely also generates R loops, leading to the mutation, but also establishes a function for the splice isoform generation and/or stabilization of ssDNA substrates for AID SRSF1-3, whose function has been hitherto unknown. Thus, our through SRSF1-3-mediated inhibition of SRSF1 specifically on findings have provided insights into both the mechanisms of the IgV genes. The AID-dependent increase of unspliced RNA AID-dependent hypermutation and the extended functions of might result from stalling and/or dissociation of elongating SR proteins in cellular metabolism. transcription complexes by the action of AID that causes DNA lesions, as initially proposed by Peters and Storb (21). Similarly, Materials and Methods disruption of cotranscriptional mRNA packaging by mutations in Analysis of Hypermutation in the IgV Gene. DT40 cells were propagated from the THO/TREX complex has been shown to promote R-loop single subclones for 1 mo. The IgVL and IgVH genes were amplified from

Kanehiro et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 genomic DNA by PCR and analyzed to find mutations. Mutations were cat- sonicated and separated by immunoprecipitation using mouse anti-AID mAb egorized as GCV or point mutation by comparing mutated sequences with (clone ZA001, Invitrogen), rabbit anti-acetylated histone H4 (Millipore), anti- published V pseudogene sequences as reported previously (49). The fre- Flag [clone M2 (Sigma-Aldrich) or clone 1E6 for quantitative PCR (Wako)], or quency of mutation events was calculated by dividing all mutation events by isotype-matched control (mouse IgG1 clone G3A1, Cell Signaling Technol- the total analyzed nucleotide numbers. An artificial GCV substrate, G/B ogy), and protein G-conjugated magnetic beads (Dynabeads Protein G; Invi- construct was also used to analyze hypermutation frequency in DT40-SW trogen Dynal AS). DNAs extracted from precipitates were analyzed by PCR. cells as described previously (39). AID expression in DT40-SW cells were switched on, and single cells expressing AID (AID-ON cells) were sorted as Analysis of Nuclear Unspliced RNA. Total RNA was extracted from the nuclear described previously (40). Colonies were transferred to 24-well plate and fraction of DT40 cells using TRIzol Reagent (Invitrogen) and treated with maintained for 1 mo between ∼1 × 105 and 1 × 106 cells. Frequency of cells with DNase I (Takara Bio). cDNA was synthesized from total RNA using random strong green fluorescence was analyzed using FACS Calibur (BD Biosciences). hexamer primers and the SuperScript II Reverse Transcriptase (Invitrogen). Spliced and unspliced IgH transcripts were amplified using KOD FX DNA SRSF1-Depletion Assay. Genomic instability caused by depletion of SRSF1 in polymerase (Toyobo). DT40-ASF cells was assessed using Tet or a Tet analog, Dox (Sigma-Aldrich) Primers used are listed in Table S2. Detailed experimental procedures are (32). After 1μg/mL Tet or 50 ng/mL Dox treatment for 2 d, DT40-ASF cells described in SI Materials and Methods. were seeded on 96-well plates at 2, 20, 200, 2,000, or 20,000 cells/well. Cells were maintained in the presence of 1μg/mL Tet or 50 ng/mL Dox, re- ACKNOWLEDGMENTS. We thank N. Matsumoto for technical assistance, Y. Kondo for chicken experiments, and J. M. Beurstedde and H. Arakawa for spectively, for 10 d, and the surviving clones were scored. plox-bsr and pExpress vectors. This work was supported, in part, by grants from the Ministry of Education, Culture, Sports, Science and Technology and ChIP. ChIP assays were carried out according to a recently reported method the New Energy and Industrial Development Technology Organization of with modifications (6). After cells were fixed and lysed, nuclear fractions were Japan. J.L.M. acknowledges support from the National Institutes of Health.

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