MBD4 Facilitates Immunoglobulin Class Switch Recombination

Abstract

Immunoglobulin heavy chain class switch recombination (CSR) requires targeted formation of DNA double-strand breaks (DSBs) in repetitive switch region elements followed by ligation between distal breaks. The introduction of DSBs is initiated by activation-induced cytidine deaminase (AID) and requires base excision repair (BER) and mismatch repair (MMR). The BER enzyme methyl-CpG binding domain protein 4 (MBD4) has been linked to the MMR pathway through its interaction with MutL homologue 1 (MLH1). We find that when Mbd4 exons 6 to 8 are deleted in a switching B cell line, DSB formation is severely reduced and CSR frequency is impaired. Impaired CSR can be rescued by ectopic expression of Mbd4 Mbd4 deficiency yields a deficit in DNA end processing similar to that found in MutS homologue 2 (Msh2)- and Mlh1-deficient B cells. We demonstrate that microhomology-rich S-S junctions are enriched in cells in which Mbd4 is deleted. Our studies suggest that Mbd4 is a component of MMR-directed DNA end processing.

Keywords: B cells; Ig class switch; Ig class switch recombination; mismatch repair; uracil glycosylase.

FIG 1

Expression of MBD4 full-length and short isoforms is lost in Mbd4-deficient CH12 cells. (A) NCBI browser screenshot of the genomic Mbd4 locus and a segment of the Ift122 gene. Mbd4 transcripts are indicated with exons (dark green boxes), untranslated regions (light green boxes), alternative transcripts (red and purple boxes), and introns (lines). (B, C, and E) Western blot analyses of MBD4 protein expression were performed using an antibody against MBD4 (directed against residues in exon 7) and nuclear extracts from WT and Mbd4^Δ2-5/Δ2-5 splenic B cells activated with LPS plus IL-4 for 48 h (B), CH12 control (Ctrl) and Mbd4 KO (1A-12^Δ/Δ) cells induced by CIT for 24 h (C), and 1A-12^+/Δ cells induced by CIT for 24 h (E). The loading control was developed with anti-lamin B1 or anti-β-actin. (B) Arrows indicate MBD4 full-length (∼70-kDa), short-form (∼18-kDa) (*), or nonspecific (NS) bands. The dashed line indicates cropping. (C) Control and 1A-12^Δ/Δ samples are from two independent experiments. (D) Mbd4 transcripts from control and KO (1A-12^Δ/Δ) cells at 0, 24, and 40 h of CIT treatment were analyzed by qRT-PCR using primers F6.1 and R1 in exon 6 and the 3′ UTR, respectively. Mbd4 transcript levels were normalized to those for 18S rRNA. The averages from two samples and two independent experiments are shown with SEMs. Asterisks indicate significant differences by Student's two-tailed t test (*, P < 0.05; **, P < 0.001). (E) Western analysis of MBD4 protein expression in CH12 (Ctrl), 1A-12^Δ/Δ, and 1A-12^Δ/+ cells.

FIG 2

CSR is impaired in Mbd4 KO cells. Control (Ctrl) CH12 cells and Mbd4-deficient cells were treated with CIT for 24 h (A, C, and D) or as indicated (B). (A) Ctrl (+/+), Mbd4 HET (1A-12^N/+, 1A-12^Δ/+, M19^N/+), and KO (1A-12^Δ/Δ) cells were analyzed by FACS for IgA expression. The Mbd4 targeted allele with (N) and without (Δ) the neo^r cassette is shown. Representative FACS analyses (left) and average percentage of IgA⁺ cells after CSR with SEMs from two to four independent experiments (except for M19^N/+ [n = 1]) (right) are shown. Asterisks indicate significant differences by Student's two-tailed t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). n.s., not significant. (B) Aicda (AID) and GLTs μ and α were analyzed by qRT-PCR, and results were normalized to those for 18S rRNA. Averages with SEMs from two samples and two independent experiments are shown. Unstim, unstimulated. (C) Western blot analysis of nuclear extracts from Ctrl and Mbd4 KO (1A-12^Δ/Δ) cells using the indicated Abs. The blot shown is representative of two independent experiments. (D) Proliferation was monitored by CFSE dilution over time in unstimulated or CIT-treated Ctrl (red) and Mbd4 KO (green) cells that were either left unstained or CFSE loaded (15 μM) and analyzed by FACS at 3 and 24 h postloading. Results shown are representative of three independent experiments.

FIG 3

Expression of truncated MBD4 has no deleterious effects on CSR. (A) Diagram of the Mbd4 locus (chromosome 6, coordinates 115840698 to 115853371 [mm9]) with a partial representation of the antisense lncRNAs (NCBI Nucleotide database accession no. CN781668 and BY200253). Splicing of Mbd4 mRNA produces Mbd4 exons 1 to 8 (XM_006505679.2) (solid line) or Mbd4 exons 1 to 3 (AK148171) (dashed line). Splicing of lncRNA exons 1* to 4′ and 1′ to 4′ (open rectangles) produces lncRNA1 (CN781668) (long dashed lines) and lncRNA2 (BY200253) (short dashed lines). PCR primers (arrowheads) are indicated. (B to E) Control (Ctrl) and Mbd4 KO (1A-12^Δ/Δ) cells were activated with CIT for 24 h. (B) Mbd4 gene expression in control and Mbd4 KO cells was examined using Affymetrix mouse genome 1.0 ST arrays (GEO accession no. GSE51559). Microarray probes hybridizing to the 5′ UTR (5U1 and 5U2), exons 1 to 5 (E1 to E5) (filled bars), exons 6 to 8 (E6 to E8), and the 3′ UTR (3U) (open bars) are indicated. (C) RT-PCR using control and Mbd4 KO cDNA templates and primer F1 in combination with primer R1, R2, or R3 detects Mbd4 exons 1 to 8, 1 to 3, or 1 to 5, respectively. Primers F4 and R4 detect lncRNAs. (D and E) Control cells were stably transfected with empty (E), MBD4 exon 1 to 5 (1-5), C-terminally MYC-tagged MBD4 exon 1 to 5 (1-5-MYC), MBD4 exon 1 to 3 (1-3), or C-terminally MYC-tagged MBD4 exon 1 to 3 (1-3-MYC) constructs. (D) Western blot analyses of nuclear extracts from stably transfected CH12 cells using anti-MBD4 or antiactin Abs. Results representative of two independent analyses are shown. (E) Average IgA switching frequencies from FACS analyses with SEMs (n = 4).

FIG 4

Complementation of impaired CSR in Mbd4-deficient cell lines. (A) The gRNA with protospacer adjacent motif (PAM) used to target Mbd4 by CRISPR-Cas9 is aligned to the genomic map of the Mbd4 gene exons (rectangles) and introns (lines). (B) The gRNA and Cas9 nuclease were transfected into CH12 control cells, and AS23 was isolated. The sequencing chromatogram of the AS23 clone depicts a homozygous 299-bp deletion spanning Mbd4 exon 8 and extending into the 3′ UTR. (C to H) CH12 control (Ctrl) and AS23 cells were either left unstimulated or treated with CIT for 20 h. (C) qRT-PCR was performed using cDNA from control and AS23 cells and primers F6 and R1 (Fig. 3A). (D) RT-PCR using control and AS23 cDNA and primer F1 in combination with primer R2, R3, or R5 detects Mbd4 exons 1 to 3, Mbd4 exons 1 to 5, or Mbd4 exons 1 to 7, respectively (Fig. 3A). (E) Proportions of control and AS23 cells expressing surface IgA. Averages and SEMs from two to four independent experiments are shown. Asterisks indicate significant differences by Student's two-tailed t test (***, P < 0.001). (F) Transcript levels for AID and for GLTs μ and α were analyzed by qRT-PCR and were normalized to those for 18S rRNA. Averages with SEMs from at least two samples and two independent experiments are shown. (G and H) Pools of AS23 cells stably expressing empty (E), MBD4^4-8 (M^4-8), and Mbd4^4-8Y514F (Y514F) constructs were analyzed. Stable transfectants were either left unstimulated or treated with CIT, as indicated. (G) Western blot assays using an anti-MBD4 or anti-lamin B1 Ab and stable AS23 transfectants. (H) Stable AS23 transfectants were analyzed for IgA surface expression by FACS analyses. Averages with SEMs from at least two samples and two independent experiments are shown.

FIG 5

Sμ DSB formation is reduced in Mbd4-deficient cells. (A) Diagrammatic summary of AID- and BER-induced DSB formation. (a) AID deaminates dC to produce uracil (U) residues. An AID hot spot motif (DGYW/WRCH [D stands for G, A, or T; H stands for C, T, or A]) is shown. (b) UNG acts on U residues to produce abasic sites. (c) AP endonucleases sever the phosphate backbone to form staggered DSBs. (d) Nucleases polish overhangs to form blunt DSBs that are substrates in the LM-PCR. (B) The BER pathway creates SSBs. MutSα (MSH2/MSH6) and MutLα (MLH1/PMS2) accumulate at U-G mismatches and attract ExoI to an adjacent nick. ExoI excises sequence between nicks on opposite strands to generate a DSB. The gap can be filled in by a translesion polymerase, or the overhang can be removed by a 5′ flap endonuclease (6). (C) Schematic showing the HindIII (H3) and SacI (S1) sites, the LM-PCR locus-specific forward primer (arrow), and the Sμ probe (horizontal shaded bar), relative to Iμ, 5′ Sμ, Sμ TRs, and Sμ core TR elements. (D) FACS analyses of forward scatter and surface IgA on CH12 control cells at 0, 12, and 24 h following CIT activation. (E) LM-PCR products were derived from control (Ctrl) and Mbd4 KO cells that were either left untreated or induced with CIT for 12 h and were then analyzed by Southern blotting using the Sμ probe. Southern blots were quantified using ImageQuant software. A representative Southern blot shows 5-fold serial dilutions of Sμ-specific LM-PCR products (filled triangles). Semiquantitative PCR amplification of the Mb1 gene (29, 31, and 33 cycles) (open triangles) was used as a loading control. (F) Relative levels of DSB induction in control and Mbd4 KO cells. Data are averages from three independent samples and SEMs. The result for the control is set to 1. Asterisks indicate significant differences by Student's two-tailed t test (**, P < 0.01).

FIG 6

Similar DSB distributions in Mbd4 KO and Msh2-deficient B cells. CH12 control (Ctrl) and Mbd4 KO cells were treated with CIT for 12 h and were then analyzed by LM-PCR. Control (n = 52) and Mbd4 KO (n = 39) LM-PCR products were subjected to DNA sequence analysis to assess DSB sites. DNA sequences surrounding DSBs from WT B cells (n = 135) and from B cells deficient in AID (n = 26), Ung (n = 17), Msh2 (n = 30), or Mlh1 (n = 16) have been described previously (8, 10). P values are from χ² analyses (*, P < 0.05; **, P < 0.01). (A) DSBs occurring at GYW/WRC AID hot spot motifs. C, control. (B to D) DSB sites located at extended AID hot spots, defined as DGYW/WRCH, were assessed. DSBs derived from WT B cells (n = 39) or from B cells deficient in Msh2 (n = 17) or Mlh1 (n = 10), activated with LPS plus IL-4 for 48 h (8, 10), or DSBs from CH12 control (n = 54) and Mbd4 KO (n = 38) cells were analyzed. (B) Proportion of DSBs located at 5′ Sμ or in Sμ TRs and their localization to the AID hot spot motif or non-hot spots. (C and D) Characterization of the DSBs by nearest-neighbor analysis using the format NX (where X indicates the nucleotide at which the break occurred and N is the 5′ neighboring nucleotide) with their localization to AID hot spot (blue) or non-hot spot (red) motifs. The percentage of breaks detected at each dinucleotide is shown.

FIG 7

Increased microhomology in Sμ-Sα junctions from Mbd4 KO cells. Sμ-Sα junctions were amplified from genomic DNA prepared from CH12 control (Ctrl) (n = 34) and Mbd4 KO (n = 24) cells stimulated with CIT for 24 h, and the DNA sequences were derived (Fig. S6 and S7). (A) Representative examples of Sμ-Sα junctions in control cells with blunt, 2-base overlap, and 8-base microhomologies (red letters). (B) Percentages of Sμ-Sα junctions with the indicated nucleotide overlaps or insertions. Chi-square analysis was used to determine P values (*, P < 0.05). (C) Mann-Whitney analysis of a box-and-whisker plot comparing the proportions of Sμ-Sα junctions displaying microhomology from CH12 control and Mbd4 KO cells.