Role for Msh5 in the regulation of Ig class switch recombination

Abstract

Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5, a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/lpr mice carrying a congenic H-2(b/b) MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long microhomologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the H-2(b) haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4-null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway.

Fig. 1.

Serum IgG3 deficiency, Msh5 gene expression, and CSR in H-2^b/b congenic MRL/lpr mice. (A) Map of the 129/Sv congenic interval in F₉ and F_≥20 congenic H-2^b/b MRL/lpr mice. The microsatellite markers and gene polymorphisms used to characterize the introgressed region are shown. (B) Serum IgG3 levels in the F₉ H-2^k/k, H-2^b/k, and H-2^b/b MRL/lpr mice. n = 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-2^k/k MRL/lpr mice and IgG^pos and IgG^neg H-2^b/b MRLlpr congenic mice (n = 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 CD19⁺ IgG3 positive cells from IgG3^pos H-2^k/k and IgG3^neg H-2^b/b MRL/lpr mice. Numbers shown are average percentage ± SEM switched cells for three mice in each group. (E) IgG3^pos H-2^k/k and IgG3^neg H-2^b/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 ± SEM; n = 10 in each group. (F) Msh5 expression profile in BALB/c (H-2^d) (n = 4), 129/Sv (H-2^b) (n = 2), C57BL/6 (H-2^b) (n = 3), and FVB (H-2^q) (n = 3) mice, using quantitative PCR (mean ± SEM). (C and F) Data represent relative Msh5 mRNA copy numbers when compared with resting B cells from H-2^k/k MRL/lpr mice (H-2^k/k MRL/lpr = 100%; mean ± SEM). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. P values were calculated by using two-tailed Student's t tests.

Fig. 2.

Increased microhomology at Sμ-Sγ3 junctions in IgG3^neg H-2^b/b congenic MRL/lpr, Msh5^−/− FVB, and Msh4^−/− C57BL/6 mice. S joints were amplified from three 6- to 8-week-old mice in the H-2^k/k MRL/lpr, IgG3^pos H-2^b/b MRL/lpr, wild-type FVB, Msh5^−/− FVB, and wild-type C57BL/6 groups, and four 6- to 8-week-old mice in the IgG3^neg H-2^b/b MRL/lpr and Msh4^−/− C57BL/6 groups. Each dot represents the number of nucleotides of donor/acceptor identity at the junction for an individual S joint. P values were calculated by using two-tailed Mann–Whitney tests.

Fig. 3.

MSH5 L85F/P786S variant has reduced binding affinity to MSH4. Yeast two-hybrid assays were performed to assess the ability of the wild-type MSH5 and MSH5 L85F/P786S variant proteins, fused with either a LexA DNA binding domain (BD) or a Gal4 activation domain (AD) to interact with wild-type MSH4. The strength of interaction was measured by using a liquid β-galactosidase assay. Data represent mean ± SE of nine replicates from three independent experiments for the AD-MSH5 L85F/P786S interaction with BD-MSH4, and 12 replicates for all other conditions in four independent experiments. Western blots of whole yeast lysates confirmed equivalent expression of the wild-type and MSH5 L85F/P786S proteins (SI Fig. 17).

Fig. 4.

Extended microhomology at B cell Ig switch joints of CVID patients carrying associated alleles of MSH5. (A) Distribution of microhomology length in Sμ-Sα1 junctions from CVID patients and controls. Each dot represents the length of microhomology of an independent S joint. The unlabeled group of controls lack any MSH5 nonsynonymous or DR3 alleles. MSH5 (L85F/P786S), heterozygote for the L85F/P786S allele. DR3++, homozygous for the rs3131378 SNP on the extended B8-DR3 MHC haplotype. TACI*, carrying one or more TACI missense mutations. Red, 0–1 bp microhomology; blue, 2–7 bp microhomology; orange, ≥ 8 bp microhomology. P values were calculated by using Mann–Whitney tests. (B) The percentage of S joints where there was “in-frame” alignment of pentamer repeat units between germline Sμ and Sα1 is represented. n = number of joints sequenced and examined. Statistical significance was determined by using two-tailed Fisher's exact tests.