The IgH Eµ-MAR regions promote UNG-dependent error-prone repair to optimize somatic hypermutation

Abstract

Intoduction: Two scaffold/matrix attachment regions (5'- and 3'-MARs_Eµ ) flank the intronic core enhancer (cEµ) within the immunoglobulin heavy chain locus (IgH). Besides their conservation in mice and humans, the physiological role of MARs_Eµ is still unclear and their involvement in somatic hypermutation (SHM) has never been deeply evaluated.

Methods: Our study analyzed SHM and its transcriptional control in a mouse model devoid of MARs_Eµ , further combined to relevant models deficient for base excision repair and mismatch repair.

Results: We observed an inverted substitution pattern in of MARs_Eµ -deficient animals: SHM being decreased upstream from cEµ and increased downstream of it. Strikingly, the SHM defect induced by MARs_Eµ -deletion was accompanied by an increase of sense transcription of the IgH V region, excluding a direct transcription-coupled effect. Interestingly, by breeding to DNA repair-deficient backgrounds, we showed that the SHM defect, observed upstream from cEµ in this model, was not due to a decrease in AID deamination but rather the consequence of a defect in base excision repair-associated unfaithful repair process.

Discussion: Our study pointed out an unexpected "fence" function of MARs_Eµ regions in limiting the error-prone repair machinery to the variable region of Ig gene loci.

Keywords: B cell; MARs region; UNG; immunoglobulin gene; somatic hypermutation (SHM).

Figure 1

MARs_Eµ deletion supports efficient in vivo Ig isotype production and GC B cell development. (A) Schematic representation of wt and MARs_Eµ ^Δ alleles (top). Targeting construct and Southern blot performed on recombinant ES cells with Neo^R insertion. Hybridization with the 5′ probe detected 4 kbp and 5 kbp SacI genomic fragments respectively for wt and recombined alleles. Hybridization with the 3′ probe detected 8 kbp and 6 kbp BamHI genomic fragments respectively for wt and recombined alleles. MARs_Eµ ^Δ allele preserved the cEµ enhancer after Cre-recombination. (B) Comparison of Peyer’s patch B cells subsets from wt and MARs_Eµ ^Δ/Δ animals by flow cytometry: dot plots showed percentage of naïve (B220⁺/GL7^-) and GC (B220⁺/GL7⁺) B cells (left panels) and, for each subset, the percentage of dividing cells (Ki67⁺) was indicated on cell count histogram plots (right panels). Experiments were performed twice with a minimum of 3 mice per group. (C) Immunoglobulin isotype secretion in sera from wt and MARs_Eµ ^Δ/Δ mice determined by ELISA (n=9 to 12 mice, mean ± SEM).

Figure 2

MARs_Eµ deletion impairs the overall SHM frequency and distribution within the IgH J-C intronic region. (A) Location of IgH regions (thick purple lines) tested for SHM, arrows represent primers used for PCR amplification. (B) Pie charts represent distribution of mutated sequences (proportional to the area in each slice, data obtained by Sanger and GS Junior sequencing method) quantified in wt and MARs_Eµ ^Δ/Δ mice in individually recombined IgH alleles. For each genotype number of individual clones is indicated in the center (after removal of clonally related sequences based on VDJ junction) and overall mutation frequencies (mutation per 1000 bp in mutated clones) are indicated below. Left: SHM downstream from J_H3 and J_H4 segments in Peyer’s patch sorted GC B cells, data obtained after cloning and sequencing by classical Sanger method. Middle: SHM downstream from J_H4 segments in spleen GC B cells sorted from SRBC-immunized mice, data obtained by NGS (GS Junior). Right: SHM downstream from cEµ region from Peyer’s patch GC sorted B cells, data obtained by classical Sanger method. (C) Graphical representation of SHM frequency in wt and MARs_Eµ ^Δ/Δ mice, quantified by NGS (Ion Proton) submitted to DeMinEr filtering, a pipeline that identifies substitution frequency at each nucleotide based on an Aicda^Δ/Δ control sample (28). Since no indication in sequence distribution is available using this method, data were represented as scattered plots, each point refers to a mutation frequency from one individual mice, mean mutation frequencies are indicated above. p-value was determined with two tailed Mann Whitney test; significant differences are indicated by: **P < 0.01 and error bars represent SEM of two independent experiments. (D) Mutation distribution along the J_H4 intron in wt (top) and in MARs_Eµ ^Δ/Δ (bottom).

Figure 3

MARs_Eµ deletion impairs strand-specific transcription upstream from Eµ region. (A) IgH locus specifying location of q-PCR probes (A, B) used for transcripts quantification. (B) Total primary transcripts quantified downstream from the J_H4 segment with probe A in Peyer’s patch GC B cells (dark colors) and in vitro-activated B cells (light colors) from wt and MARs_Eµ ^Δ/Δ mice. (C) Detection of sense transcripts (dotted arrows) in murine IgH locus (not to scale). Arrows indicate primers (S1, S2, S3) downstream from J_H3 and J_H4 used for strand-specific reverse transcription. Primary sense transcripts were quantified with probe A (black bar) in Peyer’s patch GC B cells and in vitro-activated B cells from wt and MARs_Eµ ^Δ/Δ mice. Dots indicate antisense transcript start sites according to Perlot et al. (38). Baseline levels were defined by using a RT template performed without primers (P-). Bar graphs show the relative quantity of sense transcripts obtained from template S1, S2 and S3 (mean ± SEM) from two to three independent experiments. (D) Intracellular IgM mean fluorescence intensity measured by flow cytometry in GC B cells from Peyer’s patches of wt and MARs_Eµ ^Δ/Δ mice. Bar graphs indicate data from individual mice (n=6 mice in 2 independent experiments, mean ± SEM); a representative example of cell count overlay is shown. (E) Detection of antisense transcripts (dotted arrows) in murine IgH locus (not to scale). Arrows indicate primers (AS1, AS2, AS3) downstream from J_H3 and J_H4 used for strand-specific reverse transcription. Primary antisense transcripts were quantified with probe A (black bar) in Peyer’s patch GC B (dark colors) cells and in vitro-activated (light colors) B cells from wt and MARs_Eµ ^Δ/Δ mice. Dots indicate antisense transcripts start sites according to the Alt study (38). Baseline levels were defined by using a RT template performed without primers (P-) or by using a strand-specific template that cannot be detected with A probe (T-). (F) Total primary transcripts quantified downstream from cEµ region with probe B in Peyer’s patch GC B cells (dark colors) and in vitro-activated B cells (light colors) from wt and MARs_Eµ ^Δ/Δ mice. p-value was determined with two tailed Mann Whitney test; significant differences are indicated by: *P < 0.05; **P < 0.01 and error bars represent SEM of two to three independent experiments.

Figure 4

MARs_Eµ deletion impedes error-prone repair pathways upstream from Eµ region. Comparison of IgH SHM events occurring on both sides of cEµ in Peyer’s patch GC B cells sorted from wt and MARs_Eµ ^Δ/Δ mice models, bred in genetic backgrounds deficient for base excision repair (Ung KO) and mismatch repair (Msh2 KO). Data were obtained by NGS (Ion Proton) combined to DeMinEr filtering (28). In each region, analyzed and represented as a panel, bar graphs report overall mutation frequencies (left) and detailed mutation frequencies at all bases (right). (A) Location of IgH regions (thick purple lines) tested for SHM, arrows represent primers used for PCR amplification. (B) SHM downstream from J_H4 in double-deficient Ung^Δ/Δ Msh2^Δ/Δ background. (C) SHM downstream from J_H4 in DNA repair proficient (Ung^+/+ Msh2^+/+ ) background. (D) SHM downstream from J_H4 in Ung^Δ/Δ background. (E) SHM downstream from cEµ in double-deficient Ung^Δ/Δ Msh2^Δ/Δ background. (F) SHM downstream from cEµ in DNA repair proficient (Ung^+/+ Msh2^+/+ ) background. (G) SHM downstream from cEµ in Ung^Δ/Δ background. p-value was determined with two tailed Mann Whitney test; significant differences are indicated by: *P < 0.05; **P<0.01 and error bars represent SEM of two to three independent experiments.