Class Switch Recombination Efficiency and Junction Microhomology Patterns in Msh2-, Mlh1-, and Exo1-Deficient Mice Depend on the Presence of µ Switch Region Tandem This information is current as Repeats of September 29, 2021. Jennifer Eccleston, Carol E. Schrader, Karen Yuan, Janet Stavnezer and Erik Selsing J Immunol 2009; 183:1222-1228; Prepublished online 24 June 2009; Downloaded from doi: 10.4049/jimmunol.0900135 http://www.jimmunol.org/content/183/2/1222 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/06/25/jimmunol.090013 Material 5.DC1 References This article cites 42 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/183/2/1222.full#ref-list-1
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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
Class Switch Recombination Efficiency and Junction Microhomology Patterns in Msh2-, Mlh1-, and Exo1-Deficient Mice Depend on the Presence of Switch Region Tandem Repeats1
Jennifer Eccleston,* Carol E. Schrader,† Karen Yuan,* Janet Stavnezer,† and Erik Selsing2*
The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the S tandem repeat (STR) region (STR؊/؊ mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of STR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the STR sequences, Msh2 Downloaded from deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the STR. Furthermore, in STR؊/؊ mice, deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks, particularly in sequences where nicks are infrequent. We propose that double- stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly, when the STR region is present, deficiency of Msh2 does not lead to the http://www.jimmunol.org/ increased microhomology seen with Mlh1 or Exo1 deficiencies, suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies. The Journal of Immunology, 2009, 183: 1222–1228.
ntibody gene class switch recombination (CSR)3 occurs residues introduced by AID into S regions are thought to be sub- in activated B cells and causes a switch from IgM and strates for various DNA damage repair proteins that lead to the IgD Ab expression to IgG, IgE, or IgA expression. CSR DNA breaks needed for the recombination events involved in CSR
A by guest on September 29, 2021 changes Ab effector functions while maintaining Ag specificity by (11–13). The base excision repair protein uracil DNA glycosylase exchanging the C region gene segment (C) in an Ab gene with (UNG) 2 is involved in removing U bases from DNA. Humans and one of several other downstream C region gene segments. CSR mice having defective UNG expression show substantial reduc- occurs by an intrachromosomal deletional recombination between tions in CSR (11, 12). UNG2 activity results in abasic DNA sites switch region (S region) sequences located upstream of each Ig that are cleaved by AP endonucleases (APEX1/APEX2) to create heavy chain C region gene (1). single-stranded nicks in the DNA duplex (14). A number of proteins are important for CSR. The activation- Several studies have shown that proteins involved in nonho- induced cytidine deaminase (AID) protein is required for CSR; mologous end joining (NHEJ) play an important role in CSR (15, AID activity has been shown to convert cytosine to uracil in 16). These results suggest that many CSR events represent joining ssDNA (2–8). AID has known activity hotspots, such as WRC between blunt or nearly blunt dsDNA breaks within two different sequence motifs (W ϭ A or T and R ϭ G or A) (7, 9, 10), that are S regions. AID-induced DNA nicks that are closely spaced, per- abundant in S region tandem repeat sequences. The deoxyuridine haps due to closely spaced WRC AID motifs in S region tandem repeats, could provide the nearly blunt DNA breaks needed for *Immunology Program and Department of Pathology, Tufts University School of Medicine, Boston, MA 02111; and †Program in Immunology and Virology and De- NHEJ and CSR. Widely spaced DNA nicks in S regions would partment of Molecular Genetics and Microbiology, University of Massachusetts Med- require additional DNA processing to be converted into dsDNA ical School, Worcester, MA 01655 breaks that can be used for CSR. Received for publication January 14, 2009. Accepted for publication May 12, 2009. Mismatch repair (MMR) proteins also appear to have a role in The costs of publication of this article were defrayed in part by the payment of page CSR. Mice deficient in the individual MMR proteins Msh2, Msh6, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Mlh1, Pms2, or Exo1 all exhibit partial, but significant, reductions 1 This research was supported by National Institutes of Health Grants AI24465 (to in CSR (17–21). Although each individual MMR protein-deficient E.S.), AI23283 (to J.S.), and AI65639 (to C.E.S.) and by the Eshe Foundation. mouse has a similar reduction in CSR efficiency, the different pro- 2 Address correspondence and reprint requests to Dr. Erik Selsing, Tufts University teins have different effects on the length of microhomology at CSR School of Medicine, 150 Harrison Avenue, Boston, MA 02111. E-mail address: junctions (17–22). For instance, it has been reported that B cells [email protected] from Msh2- and Exo1-deficient mice display less switch junction 3 Abbreviations used in this paper: CSR, class switch recombination; AEJ, alternative end joining; AID, activation-induced cytidine deaminase; MMR, mismatch repair; microhomology than wild-type (WT) mice, whereas switch junc- NHEJ, nonhomologous end joining; S region, switch region; STR, S tandem re- tion microhomology in Mlh1- and Pms2-deficient B cells is in- peat; UNG, uracil DNA glycosylase; WT, wild type. creased compared with WT (17, 18, 22). These results suggest that Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 different MMR protein deficiencies can have different effects on www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900135 The Journal of Immunology 1223
the CSR joining process or on the substrates available for recom- were described previously (22, 25). The third PCR used the nested primer bination. Furthermore, other studies have suggested that Msh2 fa- set SuO.8 and S␥3O.8 (5Ј-GTCTCCAGTTCTCTCACCTC-3Ј) for the first ␥ Ј Ј cilitates S region synapsis (23) and that CSR events that occur in round and SuI.8 and S 3I.8 (5 -CACATCCTCACTTGCCACTG-3 ) for the second round of PCR. Both described rounds of S-S␥1 and S-S␥3 the absence of UNG are dependent on Msh2, although the other PCR amplification performed were 35 cycles of 94°C for 30 s, 60°C for MMR proteins have not been tested (13); these findings could 30 s, and 68°C for 4 min. All PCRs were performed using Expand long indicate CSR roles for the Msh2 protein that may not involve the template Taq and Pfu polymerase mix (Roche). PCR products were cloned entire MMR machinery. However, Msh2, Mlh1, or Pms2 defi- and sequenced as previously described (25). Sequenced switch junctions were aligned to the published sequences MUSIGCD07, MUSIGHANA, ciency all result in 2- to 5-fold reductions in CSR-associated S MUSIGCD18, D78344, and FJ389571 or to S region sequences accumu- DNA double-stranded breaks (24), suggesting that these proteins lated in previous studies (25, 26) and analyzed using the GAP (Accelrys) contribute to the processing of DNA breaks during CSR. program. A Student’s two-tailed t test was used to determine the statistical Surprisingly, unlike the partial reductions of CSR observed with difference in length of microhomology between WT and mutant mouse MMR protein deficiencies in mice with intact S regions, the strains. Msh2 protein has been found to be critical for CSR in mice that Results lack the S tandem repeat (S TR) region (25). B cells that lack Mlh1 and Exo1 are critical for class switch recombination ϳ either the Msh2 protein or the S TR sequences have 2-fold re- events in regions flanking the S tandem repeats ductions in CSR, but B cells that lack both the Msh2 protein and the STR sequences have an almost complete block in CSR (19, The importance of Msh2 for CSR events in regions flanking the 25, 26). It is not clear why Msh2 has such a strong effect on CSR S TR region could reflect a requirement for a specific individual Ϫ Ϫ Ϫ Ϫ in STR / mice. We have proposed that STR / mice have function of Msh2 or the need for Msh2 as part of the entire MMR Downloaded from lower levels of AID-induced DNA nicking sites in the JH-C in- machinery. To assess whether other MMR proteins are critical for Ϫ/Ϫ tron due to the lack of the tandem repeat sequences. Therefore, we CSR in S TR flanking sequences, we crossed S TR mice with Ϫ/Ϫ Ϫ/Ϫ hypothesize that Msh2 is required and works as part of the MMR either Mlh1 or Exo1 mice to produce double mutant Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ machinery to convert widely spaced DNA nicks into dsDNA S TR :Mlh1 and S TR :Exo1 mice and analyzed breaks for CSR (24, 25, 27). However, it is also possible that Msh2 CSR in these animals. We chose to analyze double mutants lacking Mlh1 or Exo1 because results from previous studies that examined has unique functions that are needed for CSR in the absence of the http://www.jimmunol.org/ STR region. deficiencies in these MMR proteins showed similar effects on CSR In this study we have used STRϪ/Ϫ mice to assess the effects levels but different effects on switch junction microhomologies of Mlh1 and Exo1 deficiency on CSR and to determine whether the (17, 22). Msh2, Mlh1, and Exo1 MMR proteins all exhibit similar effects on To quantify CSR in the double-mutant mice, we stimulated pu- CSR efficiency and junction microhomology. We find that indeed rified B cells to undergo CSR for 4 days in culture and then ana- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ lyzed for cell surface expression of different Ab isotypes. FACS S TR :Mlh1 and S TR :Exo1 mice show the same Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ / / large reductions in CSR as seen in STR / :Msh2 / mice. This analyses from S TR :Mlh1 B cells stimulated in culture indicates that the entire MMR machinery is important for CSR with LPS and IL-4 to induce switching to IgG1 are shown in Fig. Ϫ/Ϫ Ϫ/Ϫ by guest on September 29, 2021 events that occur in the S regions that lack tandem repeats and 1A. Double-mutant S TR :Mlh1 B cells showed severe re- supports the hypothesis that the MMR machinery converts widely ductions in IgG1 switching when compared with the WT or single spaced DNA nicks into nearly blunt DNA breaks. We suggest that mutant controls. Only 5% of WT IgG1 CSR levels could be de- Ϫ/Ϫ Ϫ/Ϫ MMR-deficient B cells exhibit deficits of the near-blunt ends tected in S TR :Mlh1 B cells. Similar to previous results Ϫ/Ϫ Ϫ/Ϫ needed for NHEJ and, therefore, an increased use of an alternative (20, 26), B cells from control S TR mice and Mlh1 mice joining pathway that uses microhomologies surrounding DNA exhibit roughly 50% of WT CSR when compared with B cells nicks to join S regions. from WT littermate controls. These results show that the Mlh1 protein is critical for IgG1 CSR events that occur in sequences Materials and Methods flanking the STR region. 4 Mouse strains Fig. 1B and supplemental Table I summarize additional anal- yses using a variety of B cell stimulation conditions and show that Mlh1Ϫ/Ϫ, Exo1Ϫ/Ϫ, and STRϪ/Ϫ mice, described previously (17, 22, 26), Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ the important role of Mlh1 in STR flanking region CSR events were crossed to generate STR / :Mlh1 / and STR / :Exo1 / mice. All animal studies have been reviewed and approved by the Tufts Medical extends across multiple CH gene isotypes. The severe reduction in Ϫ/Ϫ Ϫ/Ϫ Center Division of Laboratory Animal Medicine (Boston, MA). CSR found in STR : Mlh1 mice is similar to the reduc- tions previously shown in mice that lack both Msh2 and the STR In vitro B cell cultures for flow cytometry region (25). Splenic B cells were isolated, cultured, and analyzed by flow cytometry as We also analyzed isotype switching in mice that lack the MMR described (25). protein Exo1. FACS analyses of splenic B cells cultured with LPS Ϫ/Ϫ Ϫ/Ϫ CSR junction amplification and analysis and IL-4 are shown for S TR :Exo1 double-mutant mice along with single mutant and WT littermate controls (Fig. 1C). Splenic B cells were purified as referenced above or by negative selection Consistent with a previous report (17), we found that Exo1 protein (EasySep mouse B cell enrichment kit; Stem Cell Technologies). B cells deficiency causes a 3-fold reduction in IgG1 switching (Fig. 1C). were cultured to induce IgG3 or IgG1 switching as previously described Ϫ Ϫ Ϫ Ϫ (25) or by culturing at 5 ϫ 105 cells/ml supplemented with LPS (25 g/ml; Analyses of CSR in the double-mutant STR / :Exo1 / mice Sigma-Aldrich) for IgG3 or with LPS and IL-4 (10 ng/ml; PeproTech) for show a severe reduction in IgG1 switching to ϳ5% of WT CSR IgG1. DNA was isolated from 4- to 5-day cultures by phenol/chloroform (Fig. 1C). Fig. 1D and supplemental Table II show that the reduc- separation and ethanol precipitation. Ϫ/Ϫ Ϫ/Ϫ S-S␥1 junctions were amplified using two sets of nested PCR primers. tion in IgG1 switching found in S TR :Exo1 double mu- One PCR was described previously (25). The second PCR used a nested tants extends to multiple isotypes as well. These findings indicate primer set, SuO.8 (5Ј-GATGCTGTCTCTATTCAGTTATAC-3Ј) and a critical function for both Mlh1 and Exo1 in CSR events occur- S␥1O.8 (5Ј-TTCAGTTAGGACTCCCACAC-3Ј) for the first round and ring in the sequences flanking the STR region. SuI.8 (5Ј-GAATCTATTCTGGCTCTTCTTAAGC-3Ј) and S␥1I.8 (5Ј- TTCTGCATTACTCCCAACCTC-3Ј) for the second round of PCR. S- S␥3 junctions were amplified using three different PCRs, two of which 4 The online version of this article contains supplemental material. 1224 CLASS SWITCH DEFECTS IN Mlh1- AND Exo1-DEFICIENT MICE Downloaded from
FIGURE 1. CSR is significantly reduced in STRϪ/Ϫ:Mlh1Ϫ/Ϫ and STRϪ/Ϫ:Exo1Ϫ/Ϫ mice. Purified B cells from the mouse strains indicated were cultured in vitro for 4 days with LPS and cytokines to induce CSR to each isotype and then assayed by flow cytometry to determine the ability of B cells from each mouse strain to undergo CSR. A, Representative FACS dot plots comparing IgG1 CSR in WT, Mlh1Ϫ/Ϫ,STRϪ/Ϫ, and STRϪ/Ϫ:Mlh1Ϫ/Ϫ mice. Cells expressing surface IgG1 are indicated by gated area. B, Comparisons of CSR for various isotypes in the mouse strains are indicated. Average
WT levels of CSR for each isotype (IgG1, 24%; IgG2a, 19%; IgG2b, 20%; IgG3, 18%; and IgA, 11%) were set to 100%. CSR levels for mutant strains http://www.jimmunol.org/ are shown as an average percentage of WT CSR with error bars representing the range between experiments. Single WT, STRϪ/Ϫ, Mlh1Ϫ/Ϫ, and STRϪ/Ϫ:Mlh1Ϫ/Ϫ mice were analyzed in these experiments. For these mice, multiple independent cultures were assayed and n equals the number of cultures that were used as listed below. For the WT, STRϪ/Ϫ, and STRϪ/Ϫ:Mlh1Ϫ/Ϫ mice, n ϭ 3 for all isotypes except IgG2b, where n ϭ 2. For Mlh1Ϫ/Ϫ, n ϭ 2 for all isotypes except IgG2b, where n ϭ 1. C, Representative FACS dot plots comparing, as in A, IgG1 CSR in WT, Exo1Ϫ/Ϫ,STRϪ/Ϫ, and STRϪ/Ϫ:Exo1Ϫ/Ϫ mice. D, Comparisons of CSR for the mouse strains indicated were, as in B, the average WT levels of CSR for each isotype (IgG1, 29%; IgG2b, 20%; IgG3, 15%; and IgA, 9%) were set to 100% and CSR levels for mutant mouse strains are represented as a percentage of WT CSR. Results are from two experiments that analyzed CSR levels in single cultures from each of two WT, two STRϪ/Ϫ, four Exo1Ϫ/Ϫ, and three STRϪ/Ϫ:Exo1Ϫ/Ϫ mice. by guest on September 29, 2021 Mlh1- and Exo1-deficient mice exhibit increased frequencies of To assess whether removal of STR sequences might alter the switch junctions with long stretches of microhomology that are types of switch junctions observed in mice that lack other MMR unaffected by the removal of the STR region proteins, we compared switch junction sequences in STRϪ/Ϫ: Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Previous studies have shown that MMR-deficient mice exhibit al- Mlh1 and S TR :Exo1 mice with junctions from WT, Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ tered patterns in the structures of switch junctions generated during Mlh1 , Exo1 , and S TR mice. Switch junctions in stim- CSR (17–18, 22, 25). In WT mice, a majority of switch junctions ulated B cell populations were amplified by PCR and aligned to (94–100%) exhibit a limited amount of microhomology ranging corresponding germline S region sequences (see Materials and from zero to four nucleotides in length; longer microhomologies of Methods). five nucleotides or more are found infrequently (0–3.1%) (18, 22). Table I and Fig. 2 summarize the characteristics of switch junc- ϩ/ϩ ϩ/Ϫ ϩ/Ϫ Previous studies have also indicated that Msh2 deficiency re- tions that we found in WT Mlh1 , Mlh1 , and Exo1 lit- sults in a decrease in microhomology found at switch junctions as termate mice (collectively grouped as “WT” in Table I, Fig. 2, and compared with switch junctions from WT mice. In Msh2Ϫ/Ϫ mice supplemental Fig. 1). All of these control genotypes show similar it was reported that 89% of the microhomology-based CSR joins microhomology frequencies. Consistent with previously published displayed only one or two nucleotides of microhomology, and no analyses of switch junctions in WT mice (18, 22), these control CSR join could be identified with greater than three nucleotides of junctions are mainly blunt or have short microhomologies. Switch microhomology (22). However, the switch junction patterns from junctions with longer stretches of microhomology are detected Ϫ/Ϫ Ϫ/Ϫ STRϪ/Ϫ:Msh2Ϫ/Ϫ mice appear to be distinct from the switch more frequently in Mlh1 and Exo1 mice (Table I and sup- Ϫ Ϫ junction patterns of Msh2-deficient mice. Double-deficient plemental Fig. 1). We found that 13.9% of Exo1 / S-S␥3 junc- Ϫ Ϫ STRϪ/Ϫ:Msh2Ϫ/Ϫ mice exhibit a set of S-S␥3 switch junctions tions and 6.3% of Mlh1 / S-S␥3 junctions had five or more with long stretches of microhomology that are rare in WT mice nucleotides of microhomology as compared with 2.6% in WT con- and undetectable in Msh2Ϫ/Ϫ mice (22, 25). In STRϪ/Ϫ: trols (Table I). The patterns of S-S␥3 junction microhomology in Ϫ Ϫ Ϫ Ϫ Msh2Ϫ/Ϫ mice, 19.1% of S-S␥3 junctions have five or more Mlh1 / and Exo1 / mice are statistically different from those in nucleotides of microhomology (25). These findings suggest that, WT (Table I). In addition to an increase in the percentage of junc- for Msh2 deficient mice, the ends available for joining and the tions with five or more nucleotides of homology, there was also an joining mechanism used for CSR depend on the presence or ab- increase in the average length of the microhomologies found at the sence of STR sequences. No significant increase in microhomol- Mlh1Ϫ/Ϫ (1.7 bp) and Exo1Ϫ/Ϫ (1.9 bp) switch junctions, com- ogy was found in STRϪ/Ϫ:Msh2Ϫ/Ϫ S-S␥1 junctions compared pared with WT (0.9 bp). Analogous analyses of S-S␥1 switch with WT, suggesting the possibility that Msh2 deficiency may af- junctions were performed and produced similar results as those fect the CSR joining mechanism used in an isotype-specific obtained for S-S␥3 switch junctions (Table I and supplemental manner. Fig. 1). The Journal of Immunology 1225
Table I. Comparison of CSR junction structure in WT and mutant mouse strainsa
S-S␥3 junctions
Mouse Blunt (%) 1–4 Nucleotides (%) Ն5 Nucleotides (%) Insert (%) No. of Junctions p Valueb
WT 29 47 3 21 38 STRϪ/Ϫ 25 75 0 0 16 0.1839 Exo1Ϫ/Ϫ 25 50 14 11 36 0.0245 STRϪ/Ϫ:Exo1Ϫ/Ϫ 30 50 10 10 20 0.2404c Mlh1Ϫ/Ϫ 17 68 6 8 63 0.0084 STRϪ/Ϫ:Mlh1Ϫ/Ϫ 15 50 25 10 20 0.0076 STRϪ/Ϫ:Msh2Ϫ/Ϫ 10 57 19 14 21 0.0023
S-S␥1 junctions
Mouse Blunt (%) 1–4 Nucleotides (%) Ն5 Nucleotides (%) Insert (%) No. of Junctions p Valuec
WT 31 43 0 25 32 STRϪ/Ϫ 36 59 0 5 22 0.1539 Exo1Ϫ/Ϫ 26 48 16 10 31 0.0023 Ϫ/Ϫ Ϫ/Ϫ STR :Exo1 22 56 17 6 18 0.0191 Downloaded from Mlh1Ϫ/Ϫ 28 57 4 11 54 0.0251 STRϪ/Ϫ:Mlh1Ϫ/Ϫ 11 85 0 4 27 0.0038 STRϪ/Ϫ:Msh2Ϫ/Ϫ 37 58 0 5 19 0.5544
a CSR junctions were amplified from 1–5 individual cultures from three WT (mutant mouse littermates), four STRϪ/Ϫ:Exo1Ϫ/Ϫ, one STRϪ/Ϫ: Mlh1Ϫ/Ϫ, seven Exo1Ϫ/Ϫ, and five Mlh1Ϫ/Ϫ mice. b The significance of the difference in length of microhomology between WT and mutant mouse strains was determined using a Student’s two-tailed Ͻ
t test. Values of p 0.05 were considered statistically significant. http://www.jimmunol.org/ c Increases in microhomology are statistically significant for -␥ 1 Exo1Ϫ/Ϫ and STRϪ/Ϫ:Exo1Ϫ/Ϫ and -␥ 3 Exo1Ϫ/Ϫ CSR junctions. We believe the high p value for the increase in microhomology in STRϪ/Ϫ:Exo1Ϫ/Ϫ -␥ 3 sequences is coincidental with this sample due to the fact that the increase in longer microhomologies is partially balanced by a concurrent increase in shorter microhomologies.
A previous study from Bardwell and colleagues reported switch our findings, Bardwell at al. concluded that the lack of Exo1 in B junctions in Exo1Ϫ/Ϫ mice (17). The Exo1Ϫ/Ϫ mouse strain used cells resulted in a decreased percentage of switch junction se- by Bardwell et al. is the same that we have analyzed. In contrast to quences with long microhomologies when compared with WT mice (17). We compared the Exo1Ϫ/Ϫ switch junction data pre-
sented by Bardwell et al. (17) to the data that we obtained and by guest on September 29, 2021 found that the amount of microhomology at Exo1Ϫ/Ϫ switch junc- tions is the same. In contrast, the two studies differ in the amount of microhomology found in the WT samples. In our study, the lengths of microhomologies for WT samples are shorter than the lengths of the microhomologies found in Exo1Ϫ/Ϫ mice, whereas Bardwell et al. report that lengths of microhomologies for their WT samples were longer than that for Exo1Ϫ/Ϫ mice (17). We do not know why the WT data obtained in the two studies are differ- ent; possible differences in the genetic backgrounds, the health of the animals used, or the environmental factors of housing may have affected the results. The amount of microhomology in our WT switch junctions (Table I and Fig. 2) closely resembles the amount of microhomology that has been reported for studies in- vestigating proteins other than Exo1 (18, 22). To assess whether removal of STR sequences would alter switch junctions from Mlh1- and Exo1-deficient mice, we ampli- fied switch junctions from STRϪ/Ϫ:Mlh1Ϫ/Ϫ and STRϪ/Ϫ: Exo1Ϫ/Ϫ mice and compared these switch junctions to the junc- tions amplified from Mlh1Ϫ/Ϫ and Exo1Ϫ/Ϫ mice. Similarly as in Exo1Ϫ/Ϫ and Mlh1Ϫ/Ϫ mice, we found that an increase in length Ϫ/Ϫ Ϫ/Ϫ FIGURE 2. CSR junctions from MMR-deficient B cells display in- of microhomology at CSR junctions occurs in STR :Exo1 Ϫ Ϫ Ϫ Ϫ creased microhomology compared with WT. CSR junctions were amplified and STR / :Mlh1 / mice compared with WT (Fig. 2 and sup- and sequenced from B cells activated in vitro to class switch. Percentages plemental Fig. 1). The amount of microhomologies at STRϪ/Ϫ: of S-S␥3(A) and S-S␥1(B) junctions that are blunt, have 1–4 nucle- Exo1Ϫ/Ϫ mice switch junctions is similar to the amount observed otides (nts) of microhomology, 5 or more nucleotides of microhomology, in Exo1Ϫ/Ϫ mice (Table I). STRϪ/Ϫ:Mlh1Ϫ/Ϫ double deficiency or a nucleotide insert (where the exact junction is undefined) are shown. results in a slight increase in the amount of microhomology com- CSR junctions were amplified from 1–4 in vitro cultures for each mouse analyzed. Three WT (mutant mouse littermates), one STRϪ/ϪMlh1Ϫ/Ϫ, pared with Mlh1-deficiency alone (Table I). Overall, the Mlh1- and four STRϪ/Ϫ:Exo1Ϫ/Ϫ mice were used. Results from STRϪ/Ϫ: and Exo1- deficient mice analyzed all have increased microhomol- Msh2Ϫ/Ϫ mice were published previously (25). ogy compared with WT, and removal of the STR sequences does 1226 CLASS SWITCH DEFECTS IN Mlh1- AND Exo1-DEFICIENT MICE not appear to greatly alter the pattern of switch junctions in Mlh1- or Exo1-deficient mice. This differs from the case of Msh2-defi- cient B cells, which show reduced junctional microhomology when the cells have an intact S region but increased microhomol- ogy in cells that lack the STRs (Fig. 2 and Refs. 22 and 25). In the absence of STR sequences, Msh2, Mlh1, or Exo1 deficiencies all result in a similar increase in switch junction microhomology (Fig. 2 and supplemental Fig. 1). These results suggest that the joining mechanism used for CSR events outside the STR se- quences is similarly affected by the absence of Msh2, Mlh1, and Exo1.
Discussion The results from our studies show that STRϪ/Ϫ:Mlh1Ϫ/Ϫ and STRϪ/Ϫ:Exo1Ϫ/Ϫ mice have severe defects in CSR in vitro. These defects are equivalent to a decreased efficiency of CSR to 5% or less of WT switching for each isotype analyzed. Our studies complement a previous report that analyzed CSR in STRϪ/Ϫ: Ϫ Ϫ Msh2 / mice and demonstrate that Msh2, Mlh1, and Exo1 all Downloaded from FIGURE 3. A suggested mechanism for MMR protein function during serve a critical role in CSR events in S TR flanking sequences. CSR. In this model, the Msh2-Msh6 heterodimer recognizes U-G mis- These results show that the critical role for Msh2 in most CSR matches in switch regions and recruits the Mlh1-Pms2 heterodimer to the events in STR flanking sequences is not unique to the Msh2 mismatch. This MMR complex scans the DNA for breaks generated by protein, but that these events require multiple MMR proteins. UNG and APEX1/APEX2 activity. Exo1 is recruited onto the DNA at the Taken together, the results indicate that the three proteins cooper- nick and excises DNA in the 5Ј to 3Ј direction to the mismatch. Exo1 is
ate in a common function for CSR in STR flanking sequences, inactivated and displaced from the DNA when it interacts with the Mlh1- http://www.jimmunol.org/ and we suggest that this function is the processing of widely Pms2 heterodimer (34). Exo1 is repeatedly loaded onto the DNA and ex- spaced DNA nicks into double-strand breaks. cises DNA until the mismatch is removed. Upon mismatch removal, Mlh1- MMR proteins work together to repair nucleotide mispairings Pms2 interacts with Exo1, inactivating it. This results in a uracil residue on a stretch of ssDNA that can be removed to create a double-strand break. produced during DNA replication. MMR of single-nucleotide mis- How this ssDNA would be converted into a dsDNA break is currently matches occurs by the Msh2-Msh6 heterodimer recognizing mis- unknown, but it could be due to a combination of UNG2, APEX1/2, and/or matches, recruiting and forming a complex with the Mlh1-Pms2 MRE11 activity, all of which possess the ability to act on ssDNA uracil heterodimer, and activating mismatch excision mediated by the residues (UNG) or AP sites on ssDNA (APEX1/2 and MRE11) (40–42). Exo1 protein, which is initiated at a nearby single-strand break This model is based on the proposed mechanism of MMR (31–34). Alter- (28). The Exo1 protein hydrolyzes DNA with 5Ј to 3Ј polarity and natively, MMR activity alone may generate a double-strand break if a nick by guest on September 29, 2021 therefore can only excise mismatches that lie 3Ј to a nick on the on the opposite strand is encountered during Exo1 excision, as has been same strand (29–31). The Mlh1-Pms2 heterodimer has endonu- previously proposed (23). cleolytic activity and can nick DNA near mismatches to generate a5Ј entry site for Exo1 when a nick is present in the same DNA strand (32). Reconstitution of the MMR process in vitro has dem- cessivity in the absence of the Mlh1-Pms2 heterodimer (34). We onstrated that repair of nucleotide mismatches can occur in the propose that when Msh2-Msh6, Mlh1-Pms2, and Exo1 work to- absence of Mlh1-Pms2 when there is a DNA nick 5Ј of the mis- gether during CSR, they could be involved in converting widely match. However, the Mlh1-Pms2 heterodimer is required for repair spaced S region DNA nicks into blunt or near blunt breaks to be when the DNA nick is located 3Ј of the mismatch (33). It has also joined by NHEJ proteins. As predicted by this model, fewer been shown that the Mlh1-Pms2 heterodimer limits the processiv- double-stranded breaks are detected in MMR-deficient B cells ity of the Exo1 protein, decreasing the length of excision tracts compared with WT, and an even further reduction in S double- (34). stranded breaks is observed in STRϪ/Ϫ:Msh2Ϫ/Ϫ double-defi- We propose that the CSR MMR mechanism involved in pro- cient B cells than in either STRϪ/Ϫ or Msh2Ϫ/Ϫ B cells (24). cessing switch region DNA nicks (Fig. 3) might be similar to the We have also shown that Mlh1Ϫ/Ϫ,STRϪ/Ϫ:Mlh1Ϫ/Ϫ, mechanism of MMR during DNA replication (32, 33, 35). In this Exo1Ϫ/Ϫ, and STRϪ/Ϫ:Exo1Ϫ/Ϫ mice all exhibit increased fre- model, Msh2-Msh6 recognizes U-G mismatches introduced in quencies of switch junctions that display longer stretches of mi- switch regions by AID and recruits the Mlh1-Pms2 heterodimer to crohomology. Increased microhomology at switch junctions in these sites (27). The concomitant activity of the base excision re- Mlh1Ϫ/Ϫ and Pms2Ϫ/Ϫ B cells has been suggested to reflect in- pair proteins UNG2 and APEX1/2 on neighboring U-G mis- creased use of an alternative end-joining (AEJ) pathway that is matches would result in the generation of S region DNA nicks. The distinct from classical NHEJ and that uses microhomology (18, presence of S region DNA nicks could allow for additional nicks 22). It has also been shown that CSR occurs in patients and a to be generated surrounding Msh2-Msh6-recognized mismatches mouse B cell line with impaired DNA ligase IV function, although by the Mlh1-Pms2 heterodimer. Alternatively, activity at nearby this impairment decreases the efficiency of CSR and increases U-G mismatches by the BER protein UNG2 could generate entry switch junction microhomology (36, 37). This further supports the sites for Exo1-mediated excision (35). In the absence of Mlh1- idea that AEJ pathways can be used for CSR. Such an alternative Pms2, the remaining MMR factors would need to rely on the ac- CSR joining pathway has recently been demonstrated in condi- tivity of other DNA repair proteins, such as UNG2, to create sites tional XRCC4 deficient mice, which lack the ability to join DNA that can be used for Exo1 excision. This may lead to longer Exo1 ends by NHEJ but retain the ability to undergo CSR (38–39). The excision tracks if DNA nicks are farther away from Msh2-Msh6- increased microhomology at switch junctions found in our se- recognized mismatches and also because Exo1 has increased pro- quence analyses of Mlh1- and Exo1-deficient B cells suggest that The Journal of Immunology 1227 this alternative CSR joining pathway is used at a higher frequency areas of widely staggered DNA nicks; but, again, the MMR pro- in these cells compared with WT. cess would be abortive or abnormal. This altered MMR activity The model we outline above proposes that MMR proteins con- might cause nicks/breaks (possibly DNA breaks with long single- vert widely spaced S region DNA nicks into blunt or near-blunt stranded tails) to proceed into an alternative joining pathway, lead- ends suitable for NHEJ. In the absence of MMR activity, the de- ing to CSR junctions with longer microhomologies. creased frequency of DNA breaks that can be joined by NHEJ may Additional experiments are needed to distinguish the different lead to increases in CSR through the alternative joining pathway. explanations of CSR junction microhomology results and to better However, although use of this alternative joining pathway may understand the role of MMR proteins during CSR. Production of compensate for part of the defect in CSR during MMR deficiency, mouse strains that lack both NHEJ and individual MMR proteins it cannot compensate for the CSR defect in mice deficient in both (Msh2, Mlh1, or Exo1) could indicate whether these MMR pro- individual MMR proteins and the STR sequences. The severe teins function predominately to process CSR DNA nicks for NHEJ reduction in CSR in the double-deficient mice suggests that re- ligation or whether they might have additional roles in the CSR moval of the STR region, which reduces the number of AID process. targets, results in an insufficient level of DNA breaks necessary for either CSR joining pathway. Acknowledgments Our studies analyzing CSR in mice deficient in both individual We thank Drs. Winfried Edelmann and R. M. Liskay for graciously pro- MMR proteins and the STR region has revealed an important and viding the Exo1- and Mlh1-deficient mice. We also thank Dr. Naomi collective role of MMR proteins during CSR. We find that Mlh1, Rosenberg for critically reading the manuscript.
Exo1 and Msh2 MMR proteins all have similar effects on CSR Downloaded from events that occur in the STR flanking sequences. The double Disclosures Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ knockout mice, S TR :Mlh1 ,STR :Exo1 and The authors have no financial conflict of interest. STRϪ/Ϫ:Msh2Ϫ/Ϫ, all have similar CSR junction patterns (see Ref. 25 and Figs. 1 and 2), which suggests that these deficiencies References have similar effects on the joining pathways used to complete the 1. Stavnezer, J., J. E. Guikema, and C. E. Schrader. 2008. Mechanism and regula- few CSR events that occur. Previous reports of switch junction tion of class switch recombination. Annu. Rev. Immunol. 26: 261–292. http://www.jimmunol.org/ microhomology in MMR protein-deficient mice having intact S 2. Bransteitter, R., P. Pham, M. D. Scharff, and M. F. Goodman. 2003. 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