Role for Msh5 in the regulation of Ig class switch recombination Hideharu Sekinea, Ricardo C. Ferreirab,c, Qiang Pan-Hammarstro¨ md, Robert R. Grahame, Beth Ziembab, Sandra S. de Vriesf, Jiabin Liub, Keli Hippenb, Thearith Koeuthb, Ward Ortmannb,c, Akiko Iwahoria, Margaret K. Elliotta, Steven Offerb, Cara Skonb, Likun Dud, Jill Novitzkeb, Annette T. Leeg, Nianxi Zhaoh, Joshua D. Tompkinsh, David Altshulere, Peter K. Gregerseng, Charlotte Cunningham-Rundlesi, Reuben S. Harrisb, Chengtao Herh, David L. Nelsonj, Lennart Hammarstro¨ md, Gary S. Gilkesona, and Timothy W. Behrensb,c,k aMedical University of South Carolina, Charleston, SC 29425; bUniversity of Minnesota Medical School, Minneapolis, MN 55455; dKarolinska University Hospital, SE-141 86 Huddinge, Sweden; eBroad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02142; fThe Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands; gFeinstein Institute for Medical Research, Manhasset, NY 11030; hWashington State University, Pullman, WA 99164; iMount Sinai School of Medicine, New York, NY 10029; and jNational Cancer Institute, Bethesda, MD 20892 Communicated by Richard H. Scheller, Genentech, Inc., South San Francisco, CA, February 19, 2007 (received for review December 8, 2006) Ig class switch recombination (CSR) and somatic hypermutation by forming heterodimers; Msh2-Msh6 (MutS␣), Msh2-Msh3 serve to diversify antibody responses and are orchestrated by the (MutS␤), Msh4-Msh5 (MutS␥), Mlh1-Pms2 (MutL␣), and Mlh1- activity of activation-induced cytidine deaminase and many pro- Mlh3 (MutL␥) (3). MutS heterodimers are thought to recruit MutL teins involved in DNA repair and genome surveillance. Msh5,a heterodimers. Experiments using Mut homologue gene-knockout gene encoded in the central MHC class III region, and its obligate (KO) mice revealed that Msh2-, Msh6-, Mlh1-, and Pms2-deficient heterodimerization partner Msh4 have a critical role in regulating animals had decreased efficiency of CSR and somatic hypermuta- meiotic homologous recombination and have not been implicated tion (4). Deficiencies of MutS and MutL genes often result in in CSR. Here, we show that MRL/lpr mice carrying a congenic H-2b/b differences in microhomology lengths at S joints and show three MHC interval exhibit several abnormalities regarding CSR, includ- phenotypes, decreased (Msh2Ϫ/Ϫ and Mlh3Ϫ/Ϫ) (4, 5), no change ing a profound deficiency of IgG3 in most mice and long micro- (Msh6Ϫ/Ϫ) (6), or increased (Mlh1Ϫ/Ϫ and Pms2Ϫ/Ϫ) (5, 7) micro- homologies at Ig switch (S) joints. We found that Msh5 is expressed homology. The differences in S joint phenotypes between Msh2Ϫ/Ϫ at low levels on the H-2b haplotype and, importantly, a similar long mice and Mlh1Ϫ/Ϫ or Pms2Ϫ/Ϫ mice suggest the existence of other S joint microhomology phenotype was observed in both Msh5 and proteins that function in the same pathway of CSR as Mlh1 and Msh4-null mice. We also present evidence that genetic variation in Pms2. MSH5 is associated with IgA deficiency and common variable Msh5 and Msh4 are involved in the resolution of DNA Holliday immune deficiency (CVID) in humans. One of the human MSH5 junctions, the four-stranded DNA structures that form during alleles identified contains two nonsynonymous polymorphisms, homologous recombination in meiosis (8). Msh4 and Msh5 KO and the variant protein encoded by this allele shows impaired mice are sterile due to an inability to resolve these meiotic chro- binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA mosomal crossovers (9–11). deficiency patients carrying disease-associated MSH5 alleles show Based on these studies in mice, the Mut homologues are attrac- increased donor/acceptor microhomology, involving pentameric tive candidate genes for human Ig deficiencies. Selective IgA DNA repeat sequences and lower mutation rates than controls. Our deficiency (IgAD) (serum IgA Ͻ0.05 g/liter) is the most common findings suggest that Msh4/5 heterodimers contribute to CSR and primary immunodeficiency disorder in man, with a prevalence of support a model whereby Msh4/5 promotes the resolution of DNA Ϸ1/600 Caucasian individuals (12). The selective nature of the CSR breaks with low or no terminal microhomology by a classical defect in IgAD is not understood. Common variable immune nonhomologous end-joining mechanism while possibly suppress- deficiency (CVID) is a more severe disease and affects Ϸ1/25,000 ing an alternative microhomology-mediated pathway. Caucasians. Patients show a marked reduction in serum levels of both IgG (usually Ͻ3 g/liter) and IgA (Ͻ0.05 g/liter), together with ͉ ͉ immunoglobulin subclass deficiency mismatch repair Msh4 reductions of IgM in about half the cases (Ͻ0.3 g/liter). CVID patients have a high incidence of infectious complications and, fter appropriate stimulation, B cells undergo class switch paradoxically, are prone to autoimmune disorders (13). Arecombination (CSR), whereby the functionally rearranged The available evidence suggests a common genetic basis for V(D)J DNA segment is recombined with a downstream Ig constant IgAD and CVID (14) and individuals with IgAD may transition region segment. The biochemistry of CSR is complex and involves into CVID. Haplotypes of the MHC show genetic association with the B cell-specific gene activation-induced cytidine deaminase, IgAD, notably HLA (HLA) A1-B8-DR3 and B14-DR1 (15–17). which initiates both CSR and somatic hypermutation (1). CSR also requires many ubiquitously expressed genes important for detecting DNA mismatches and breaks and regulating DNA repair (2). CSR Author contributions: H.S. and R.C.F. contributed equally to this work; H.S., R.C.F., L.H., occurs at specific DNA segments called switch (S) regions, which lie G.S.G., and T.W.B. designed research; H.S., R.C.F., B.Z., J.L., K.H., T.K., A.I., M.K.E., S.O., C.S., L.D., N.Z., and J.D.T. performed research; Q.P.-H., S.S.d.V., J.N., A.T.L., D.A., P.K.G., C.C.-R., upstream of each constant region and contain hotspots for activa- C.H., D.L.N., and L.H. contributed new reagents/analytic tools; H.S., R.C.F., Q.P.-H., R.R.G., tion-induced cytidine deaminase-mediated cytosine deamination. B.Z., J.L., K.H., W.O., R.S.H., G.S.G., and T.W.B. analyzed data; and H.S., R.C.F., G.S.G., and IMMUNOLOGY The ligation of the S␮ region with the downstream S regions is T.W.B. wrote the paper. carried out by protein factors that comprise the nonhomologous The authors declare no conflict of interest. end joining machinery for DNA repair (1, 2). Abbreviations: CSR, class switch recombination; CVID, common variable immune defi- Mismatch repair proteins play a critical role in safeguarding ciency; IgAD, IgA deficiency; KO, knockout. genetic stability. The key proteins for initiation of eukaryotic cPresent address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. mismatch repair are homologues of bacterial MutS and MutL. In kTo whom correspondence should be addressed. E-mail: [email protected]. mammals, there are five MutS (Msh2, Msh3, Msh4, Msh5, and This article contains supporting information online at www.pnas.org/cgi/content/full/ Msh6) and four MutL (Mlh1, Mlh3, Pms1, and Pms2) homologues. 0700815104/DC1. Each Mut homologue acts at the DNA repair or recombination site © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700815104 PNAS ͉ April 24, 2007 ͉ vol. 104 ͉ no. 17 ͉ 7193–7198 Downloaded by guest on September 28, 2021 b/b Fig. 1. Serum IgG3 deficiency, Msh5 gene expression, and CSR in H-2 congenic MRL/lpr mice. (A) Map of the 129/Sv congenic interval in F9 and FՆ20 congenic H-2b/b MRL/lpr mice. The microsatellite markers and gene polymorphisms used to characterize the introgressed region are shown. (B) Serum IgG3 levels in the k/k b/k b/b F9 H-2 , H-2 , and H-2 MRL/lpr mice. n ϭ 12–16 mice in each group at 12 weeks of age. The number of mice in each group decreased with aging because of mortality. Bars indicate mean values. (C) Msh5 mRNA expression levels were measured in cDNA from splenic B cells of H-2k/k MRL/lpr mice and IgGpos and IgGneg H-2b/b MRLlpr congenic mice (n ϭ 3 each) (D) CSR of splenic B cells was induced in vitro with LPS for class switch induction to IgG3. Representative FACS plots show the percentage of CD19ϩ IgG3 positive cells from IgG3pos H-2k/k and IgG3neg H-2b/b MRL/lpr mice. Numbers shown are average percentage Ϯ SEM switched cells for three mice in each group. (E) IgG3pos H-2k/k and IgG3neg H-2b/b MRL/lpr mice were immunized with TNP-LPS or TNP-Ficoll, and IgG2b (Right) and IgG3 (Left) anti-TNP responses were measured at 2 weeks. Serum OD380 values are represented on the y axis. Data shown represent the mean Ϯ SEM; n ϭ 10 in each group. (F) Msh5 expression profile in BALB/c (H-2d)(n ϭ 4), 129/Sv (H-2b)(n ϭ 2), C57BL/6 (H-2b)(n ϭ 3), and FVB (H-2q)(n ϭ 3) mice, using quantitative PCR (mean Ϯ SEM). (C and F) Data represent relative Msh5 mRNA copy numbers when compared with resting B cells from H-2k/k MRL/lpr mice (H-2k/k MRL/lpr ϭ 100%; mean Ϯ SEM). *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. P values were calculated by using two-tailed Student’s t tests. Homozygosity for the A1-B8-DR3 haplotype is a particularly strong antibody phenotypes were similar in congenic H-2b/b MRL/lpr risk factor for IgAD in Caucasians, with an incidence reported as animals backcrossed nine generations, and those animals back- high as 13% (18).