Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation

Abstract

Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil-DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung(-/-) mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung(-/-) mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung(-/-) mice depends upon the endogenous SMUG1 uracil-DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung(-/-) Smug1(-/-) mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2-dependent mechanism, Ung(-/-) Smug1(-/-) mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung(-/-) background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.

Keywords: Class switching; DNA deamination; Somatic hypermutation; Uracil.

Figure 1

SMUG1 deficiency on its own has no effect on immunoglobulin CSR. (A) The proportion of B cells expressing surface IgG1 from Smug1^−/− (gray bars) and littermate Smug1^+/− (open bars) mice was monitored by flow cytometry over 5 days of culture after in vitro stimulation with LPS and IL‐4. Data show mean ± SEM from eight mice representative of three independent experiments (two‐tailed unpaired t‐test). (B) Serum immunoglobulin IgM and IgG proteins were purified on protein L and separated by SDS/PAGE, using n = 6 control mice and n = 5 Smug1^−/− mice at 6 months of age. Arrows indicate the mobility of κ, kappa light chains, γ, IgG, and μ, IgM heavy chains.

Figure 2

The age‐dependent partial compensation of CSR in UNG‐deficient mice is compromised in SMUG1/UNG double‐deficient mice. (A) Serum from 6‐month‐old Smug1^−/−Ung^−/− mice (n = 5) and Ung^−/− mice (n = 7) was assessed for the presence of immunoglobulin chains by immunoprecipitation on protein L and gel electrophoresis. Arrows indicate the mobility of κ, kappa light chains, γ, IgG, and μ, IgM heavy chains (B, C). Immunoelectrochemiluminescent quantification of (B) IgM and (C) IgG3, IgG1, IgG2b, and IgA (in μg/mL) in serum collected at 26 weeks from mice of different uracil excision genotypes. Each symbol represents one mouse, with the mean (horizontal bars ± SEM) indicated above each group. Labels denote genotype: S⁺U⁺: Smug1⁺ Ung⁺ control mice; S: Smug1^−/−; U: Ung^−/−; SU: Smug1^−/−Ung^−/−; AID: Aicda^−/−. (D) Switched isotype titers were measured as in (C), using serum from mice at 13 weeks of age. (E) Changes in average titers of switched serum isotypes over time in Ung^−/− versus Smug1^−/−Ung^−/−. Values taken from (C) and (D) are expressed as a fraction of the mean titers in uracil excision‐proficient mice. (C and D: Two‐tailed unpaired t‐test was used to compare Ung^−/− and Smug1^−/−Ung^−/− mice).

Figure 3

CSR in vitro is further impaired in uracil excision null B cells. (A) The proportion of IgG1⁺ B cells was determined by surface staining and flow cytometry after 3, 5, and 7 days of culture in vitro in the presence of LPS + IL‐4. Horizontal bars show mean ± SEM, with the value from cultures from individual animals represented by a symbol. Data shown are representative of six independent experiments. (B) Splenic B cells were cultured for 5 days and the fraction of IgG1⁺ cells was determined as in (A) and expressed as a fraction of the per‐experiment average obtained from UNG‐deficient animals. Data aggregated from six independent experiments comprising a total of 50 individual animals indicated as data points. (C) Using data from (A), genotype‐dependent changes were assessed in the fraction of switched cells in culture at the three indicated time points. (D) Serum IgG1 titers (μg/mL) at 6 weeks of age were assessed by ELISA from litters born to Aicda^−/− mothers and deficient in both UNG and SMUG1 or UNG only and compared with uracil excision‐proficient controls. Each symbol represents the titer of an individual mouse with the means shown as horizontal bars ± SEM. Data are from three independent litters. (E) Sequence homology in Sμ–Sγ1 junctions PCR‐amplified from splenic B cells after 7 days of culture in LPS and IL‐4. In black, the proportion of junctions with two or more residues identical to both μ and γ1 switch regions. n indicates the number of sequences analyzed per genotype. Labels denote genotype: S⁺U⁺: Smug1⁺ Ung⁺ control animals; S: Smug1^−/−; U: Ung^−/−; SU: Smug1^−/−Ung. (A, B, and D: two‐tailed unpaired t‐test; E: Fisher's exact test).

Figure 4

SHM analysis of germinal center B cells reveals a dependence of A:T hypermutation on uracil excision. (A) Mutations observed in the J_H4 intronic region from rearranged Peyer's patches B cells. The total number of mutations from the indicated individual animals is shown for each genotype. The mutations at each base, expressed as a percent of the total, were normalized to the base composition of the region analyzed. (B) Distribution of nucleotide substitutions at G:C and A:T pairs in the J_H4 intron. Comparison of uracil excision‐proficient (control, top histogram) versus uracil excision‐deficient (Smug1^−/−Ung^−/−, bottom histogram) B cells. Each bar represents percent of total mutations. (C) The normalized ratios of A:T to C:G mutations in datasets from each individual mouse are shown grouped per genotype. Each symbol represents the value obtained for an individual mouse with bars indicating the mean ± SEM (two‐tailed unpaired t‐test). (D, E) The J_κ5 intronic region was analyzed as in (A) and (B). (F) Statistical comparison of mutation distributions using Fisher's exact test. Labels denote genotype: S⁺U⁺ and BL/6: Smug1⁺ Ung⁺ and C57BL/6J controls (ctrl), respectively; S: Smug1^−/−; U: Ung^−/−; SU, Smug1^−/−Ung^−/−.

Figure 5

Uracil recognition pathways in antibody diversification. (A) CSR. Efficient base excision by UNG (or by highly overexpressed SMUG1) of densely spaced uracil residues in the immunoglobulin switch region repeats followed by cleavage by APE1 results in frequent double‐strands breaks that can be joined by nonhomologous end joining with minimal end processing (left path). In the absence of UNG, or in regions with lower AID hotspot density (right path), nicks are typically too far apart to allow direct formation of double‐strand breaks. Class switching thus relies on processing of U:Gs by the MMR machinery (comprising the MSH2/MSH6//MLH1/PMS2 heterotetramer) to create staggered breaks, which are resolved via microhomology‐dependent alternative end joining. Even rare nicks, mostly provided by SMUG1 in the absence of UNG, could facilitate Exo1 resection by licensing of the endonuclease activity of PMS2 in the MMR complex, and thus promote the formation of double‐strand breaks leading to class switching. (B) SHM. The contribution of uracil excision to the MHS2‐dependent phase 2 (left path) is evidenced by the lowered proportion of A:T mutations observed in the absence of UNG, which is further depressed in the absence of SMUG1. The creation of an MMR‐dependent resection patch is known to be potentiated by nicks in the region surrounding the mismatch, suggesting that uracil excision facilitates phase II by providing a source of nicks. As in the case of CSR, even isolated, distal nicks could license the PMS2 endonuclease to make further incisions and promote efficient patch resection. It is unknown, but plausible, that mutagenic MMR in the absence of uracil excision by both UNG and SMUG1 exploits other sources of nicks. Subsequent to Exo1 resection, the patch is refilled by error‐prone Y‐family polymerases, among which Polη plays the dominant role for hypermutation. In the absence of MSH2 (right path), efficient uracil excision by UNG is absolutely essential for mutagenesis at A:T pairs, and Polη cannot be substituted for by other endogenous polymerases, suggesting that they cannot (with detectable efficiency) facilitate mutagenesis across the relatively short patches of BSR.