The IgH 3′ regulatory region controls somatic hypermutation in germinal center B cells

3′RR-deficient mice display normal size B cell compartments but a globally decreased Ig secretion, including a partial IgM defect and culminating in a complete blockade of classed-switched isotypes, a phenotype involving both decreased Ig secretion in plasma cells and decreased CSR frequency (Vincent-Fabert et al., 2010). Because CSR and SHM are well-known temporally and spatially associated processes, both involving AID-initiated DNA lesions (Neuberger and Rada, 2007; Schatz and Ji, 2011), we explored 3′RR-deficient mice for potential SHM defects. Mice were immunized orally with sheep red blood cells for 2 wk and intraperitoneally with 10 µg LPS for 3 d. Oral immunization with sheep red blood cells together with 3-d intraperitoneal injection of LPS corresponds to the immunization protocol that we found the most efficient to regularly obtain in vivo activated B cells in both spleen and Peyer’s patches, with high levels of somatic mutation. We are thus now routinely using this protocol as a standard for in vivo B cell stimulations. Similar occurrence (P > 0.5) of germinal center B cells (B220⁺PNA^highFas⁺) were found in Peyer’s patches of 3′RR-deficient and WT mice: 17.6 ± 3.5% vs. 18.6 ± 1.8% of PNA^highFas⁺ cells among B220⁺ cells; means obtained from groups of five mice. Numbers of Peyer’s patches were similar in 3′RR-deficient (mean of 7, range 5–10, n = 8) and WT mice (mean of 6, range 4–8, n = 8). The total number of cells in Peyer’s patches was slightly higher in 3′RR-deficient (mean of 22 × 10⁶ cells/mouse, range 13–31, n = 8) than in WT mice (mean of 8 × 10⁶ cells/mouse, range 5–10, n = 8). Rearranged IgH V(D)J regions from B220⁺PNA^highFas⁺ cells (10 3′RR-deficient and 8 WT mice) were cloned and sequenced to evaluate the role of the 3′RR in SHM. Ensuring that the reference SHM level in B220⁺PNA^highFas⁺ cells pooled from the eight WT mice was not biased by any “jackpot” high SHM in a few mice, four individually evaluated animals under this immunization protocol consistently and homogeneously showed the high SHM rate expected in WT mice (Fig. 1 B, right). A dramatic decrease (P < 0.0001, Mann–Whitney U test) in the relative number of mutated sequences was found in 3′RR-deficient mice (5%, 6/119) as compared with WT (94%, 121/129; Fig. 1 A). The mutation frequency in 3′RR-deficient mice (14 mutations among 64,044 bp analyzed, 0.022%) was compared with WT (902 mutations among 68,841 bp analyzed, 1.3%) and showed a >98% reduction (P < 0.0001, Mann–Whitney U test). When comparing the frequency of mutations per sequence, we found in 3′RR-deficient mice a lower frequency (1% compared with 57% in WT, P < 0.0001) of clones carrying multiple mutations (over five mutations in the analyzed region; Fig. 1 B, left). Although the number of mutations in 3′RR sequences is small, we found that the proportion of mutations at C/G did not significantly differ between WT (43.3%) and 3′RR-deficient mice (35.7%) and that A↔G transitions represented 50.8% and 41.0% for WT and 3′RR-deficient mice, respectively (not depicted). Thus, deletion of the IgH 3′RR nearly abrogates the SHM process in V_H regions.

We investigated the transcripts encoding several proteins involved in SHM (Neuberger and Rada, 2007). Transcripts for AID, POLη, REV1, UNG, MSH2, and MSH6 were detected in similar amounts (P > 0.1) in Peyer’s patch B cells from 3′RR-deficient and WT mice, showing that at the RNA level, 3′RR-deficient B cells normally express the SHM trans-acting machinery (not depicted). Thus, lack of SHM in IgH V(D)J segments in 3′RR-deficient mice is not related to a deficiency of AID and related cofactor expression.

To further check that the lack of SHM at the heavy chain locus is not related to a global decrease of SHM in 3′RR-deficient mouse B cells, we investigated SHM in the Ig κ locus. A similar number of mutated sequences was found in 3′RR-deficient (87%, 33/38) and WT mouse B cells (91%, 44/48; Fig. 1 A). The mutation frequency in 3′RR-deficient mice (255 mutations among 18,282 bp analyzed, 1.39%) was similar to WT (296 mutations among 24,376 bp analyzed, 1.21%). When comparing the number of mutations per sequence, we found a similar number of clones from 3′RR-deficient cells that carried multiple mutations (i.e., with over five mutations in the analyzed region: 45% for 3′RR-deficient mice compared with 52% in WT; Fig. 1 C). The proportion of transitions did not differ between WT (52.4%) and 3′RR-deficient mice (51.8%), and A↔G transitions represented 43.8% and 34.0% for WT and 3′RR-deficient mice, respectively (not depicted). Thus, deletion of the 3′RR specifically abrogates the SHM process in the heavy chain locus without significantly affecting the κ light chain locus.

SHM correlates with transcription (Fukita et al., 1998). Alterations of the SHM rate have been reported using BAC transgenes with large deletion of the 3′RR, although transcription of these transgenes was variably affected by the deletion (Dunnick et al., 2009). In contrast, the partial IgH transcription defect observed in hs3b/4-deficient mouse resting B cells did not lead to any significant SHM decrease in germinal center B cells (Morvan et al., 2003). The situation is quite different in 3′RR-deficient mice, which showed a nearly complete V(D)J SHM blockade (98% of reduction) along with only a partial decrease (40%) of IgH primary transcripts in B220⁺PNA^highFas⁺ Peyer’s patch B cells (Fig. 2 A). In contrast, deletion of the 3′RR did not affect SHM (Fig. 1 C) and transcription at the κ light chain locus (Fig. 2 B). Our model indicates that Ig heavy chain primary transcription is maintained at a fairly high level in germinal center B cells devoid of 3′RR, although SHM is almost completely abrogated. This suggests that such a strong SHM defect cannot solely result from decreased transcription. Rather, our data suggest that the 3′RR could be directly essential for the recruitment of AID onto the IgH locus, a hypothesis which fits well with recent results showing that the IgH 3′RR is itself an AID target (Péron et al., 2012). To determine whether AID recruitment to the J_H4 region is dependent on the 3′RR, we performed chromatin immunoprecipitation (ChIP) analysis using an anti-AID antibody on chromatin prepared from B splenocytes of WT, 3′RR-deficient, and AID-deficient mice (immunized orally with sheep red blood cells for 2 wk and intraperitoneally with 10 µg LPS for 3 d). We found that AID occupancy at the J_H4 region was dramatically reduced in 3′RR-deficient mice as compared with WT (Fig. 2 C). Only background levels of immunoprecipitation were found both for 3′RR-deficient and AID-deficient mice, leading to the conclusion that AID enrichment to the IgH J_H4 region is drastically impaired in 3′RR-deficient mice. Confirming results obtained with Peyer’s patches, deletion of the 3′RR lead only to a partial decrease (50%) of IgH primary transcripts in B220⁺PNA^high splenic B cells (Fig. 2 D) without significant effect on κ primary transcripts (Fig. 2 E). The significant binding of RNA polymerase II on the IgH allele of 3′RR-deficient mice (after 3-d LPS stimulation of splenic B cells in vitro) confirms that transcription is maintained (Fig. 2 F). This work provides the first conclusive evidence that, first, AID-mediated SHM is targeted to the IgH locus by the cis-acting 3′RR and, second, that SHM within Ig genes does not imply parallel transcription. These results are reminiscent of those reported for 3′ regulatory elements located in the chicken Ig light chain (Kothapalli et al., 2008; Blagodatski et al., 2009). Similarly to the IgH 3′RR, the DIVAC (diversification activator) cis-acting element is required for SHM at the chicken Ig light chain gene (Blagodatski et al., 2009) but does not function by increasing transcription (Kohler et al., 2012). Although high levels of transcription are not sufficient for robust SHM in IgL locus (Yang et al., 2006), separated mutational and transcriptional enhancers were recently reported for IgL genes (Kothapalli et al., 2011). The mechanism of how cis-regulatory sequences activate SHM in neighboring transcription units remains speculative. As hypothesized for DIVAC (Blagodatski et al., 2009), the IgH 3′RR might promote the formation of protein complexes that first bind AID and then hand it over to the neighboring transcription initiation complex.

Studies reported that S_μ targets SHM; mutational frequency peaked 5′ of S_μ and decreased thereafter (Maul and Gearhart, 2010). We investigated whether deletion of the 3′RR abrogated this pattern of SHM. Because of high GC content and the repetitive nature of the S_μ sequence (Fig. S1), accurate PCR amplification across the region is known to be problematic. We thus cloned the entire I_μ-C_μ sequence (location of PCR primers in Fig. S1) and sequenced both the 5′ and the 3′ part of the PCR product encompassing S_μ. Experiments were performed with B splenocytes of WT and 3′RR-deficient mice immunized orally with sheep red blood cells and intraperitoneally with LPS. A dramatic decrease (P < 0.0001, Mann–Whitney U test) of mutated sequences 5′ to S_μ was found in 3′RR-deficient mice (14%, 5/36) as compared with WT (65%, 20/31; Fig. 1 A). The mutation frequency in 3′RR-deficient mice (7 mutations among 19,078 bp analyzed, 0.036%) was compared with WT (92 mutations among 20,934 bp analyzed, 0.44%) and showed a >90% reduction (Fig. 1, A and D). In agreement with previous results (Maul and Gearhart, 2010), no mutations were documented 3′ to S_μ in WT nor in 3′RR-deficient mice (not depicted). ChIP experiments indicated that AID occupancy at the S_μ region was markedly reduced in 3′RR-deficient splenocytes as compared with WT (Fig. 2 G). Only background levels of immunoprecipitation were found both for 3′RR-deficient mice and AID-deficient mice, leading to the conclusion that AID binding to S_μ is totally impaired in 3′RR-deficient mice.

There have been several previous genomic alterations of the IgH 3′RR, notably through individual enhancer deletions (Manis et al., 1998; Pinaud et al., 2001; Vincent-Fabert et al., 2009; Bébin et al., 2010). Partial functional redundancies of the four 3′RR enhancers have long kept their role quite elusive, especially with regard to SHM. The complete deletion now clarifies that the 3′RR is mandatory for SHM. This major SHM defect paralleled a partly decreased IgH transcription, suggesting that elements beyond primary transcription accessibility are missing for recruitment of the SHM machinery onto the 3′RR-deficient IgH locus. The very low residual SHM suggests that no additional elements cooperate with the 3′RR for SHM. Although transcription is a prerequisite for several regulatory mechanisms controlling key stages in the B cell physiology, it is clearly not sufficient to ensure Ig locus accessibility to AID. The 3′RR-deficient mice will facilitate further elucidation of how the 3′RR “enhanceosome” or the 3′RR-dependent IgH locus 3D-conformation might regulate AID accessibility beyond transcriptional regulation.

Our research has been approved by the ethics committee review board of our university (Limoges, France) and hospital (Comité Régional d’Ethique sur l’Expérimentation Animale du Limousin, CHU Dupuytren, Limoges, France). Generation of 3′RR-deficient mice has been previously reported (Vincent-Fabert et al., 2010) and was performed on a C57BL/6 background. Mice were bred and maintained under specific pathogen–free conditions. Age-matched littermates (8–12 wk old) were used in all experiments. C57BL/6 mice were used as WT mice.

Mouse immunizations were performed orally with sheep red blood cells for 2 wk and intraperitoneally with 10 µg LPS for 3 d. Single-cell suspensions from Peyer’s patches were labeled with B220-APC–, PNA-FITC–, and Fas-PE–conjugated antibodies. Purification of B220⁺PNA^highFas⁺ cells was realized on a FACSVantage (BD). These cells were used for SHM analysis of V(D)J-rearranged fragments and Igκ light chain VJ-rearranged fragments and analysis of IgH and κ primary transcripts. Single-cell suspensions from splenocytes were labeled with B220-APC– and PNA-FITC–conjugated antibodies for purification of B220⁺PNA^high B splenocytes. These cells were used for SHM analysis of S_μ.

Genomic DNA was extracted from sorted B220⁺PNA^highFas⁺ cells. IgH V(D)J-rearranged fragments were amplified by PCR using the following primers and multistep programs: the forward primer 5′-GCGAAGCTTARGCCTGGGRCTTCAGTGAAG-3′, complementary to the VHJ558 segment, and the backward primer 5′-AGGCTCTGAGATCCCTAGACAG-3′, corresponding to a sequence 517 bp downstream of the J_H4 segment using 1 cycle at 98°C for 30 s, 33 cycles (98°C for 10 s, 67°C for 30 s, and 72°C for 90 s), and 1 cycle at 72°C for 10 min. Igκ light chain VJ-rearranged fragments were amplified by PCR as previously reported (Xiang and Garrard, 2008) using the following primers and multistep programs: the forward primer 5′-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3′ (consensus for Vκ) and the backward primer 5′-AGCGAATTCAACTTAGGAGACAAAAGAGAGAAC-3′ (found 557 bp downstream of the J_κ5 segment) using 1 cycle at 98°C for 30 s, 33 cycles (98°C for 10 s, 66°C for 30 s, and 72°C for 90 s), and 1 cycle at 72°C for 10 min. S_μ from B220⁺PNA^high B splenocytes was amplified by PCR using Herculase II DNA polymerase (Agilent Technologies) and the following primers and multistep programs: the forward primer 5′-CTCTGGCCCTGCTTATTGTTG-3′ and the backward primer 5′-GAAGACATTTGGGAAGGACTGACT-3′ using 1 cycle at 95°C for 120 s, 40 cycles (95°C for 15 s, 61°C for 20 s, and 72°C for 180 s), and 1 cycle at 72°C for 180 s. The exact location of the PCR primers and the S_μ PCR product used for mutation analysis are shown in Fig. S1.

PCR products were cloned into the Zero Blunt Topo PCR cloning vector system (Invitrogen). Plasmids were isolated using the NucleoSpin kit (Macherey-Nagel) and sequenced using an automated laser fluorescent ANA ABI-PRISM sequencer (PerkinElmer).

B cells from spleen of IgH 3′RR-deficient or WT mice were purified using CD43-coupled beads from Miltenyi Biotec according to the manufacturer’s recommendations. Total RNA was extracted using tri-reagent (Ambion). Real-time PCR was performed in duplicate by using TaqMan assay reagents and analyzed on an ABI Prism 7000 system (Applied Biosystems). Transcripts were studied for Msh2 (reference Mn00500563-m1), Msh6 (reference Mn00487769-g1), UNG (reference Mn00449156-m1), POLη (reference Mn00453168-m1), REV1 (reference Mn00450983-m1), AID (reference Mn01184115-m1), and GAPDH (used for normalization of gene expression levels; reference Mm99999915-g1) as previously reported (Truffinet et al., 2007; Vincent-Fabert et al., 2009). B220⁺PNA^highFas⁺ cells were used for analysis of IgH and κ primary transcripts. IgH primary transcripts (probe located in the intron between the last J_H and the intronic E_μ enhancer) were studied as previously reported (Tinguely et al., 2012): forward primer, 5′-TTCTGAGCATTGCAGACTAATCTTG-3′; reverse primer, 5′-CCTAGACAGTTTATTTCCCAACTTCTC-3′; and probe, 5′-CCCTGAGGGAGCCG-3′. κ primary transcripts (probe located in the intron between the last J_κ and the intronic enhancer Eiκ): κ forward primer, 5′-ACCCCCGCGGTAGCA-3′; κ reverse primer, 5′-TCCTATCACTGTGCCTCAGGAA-3′; and probe, 5′-CCCTTGCTCCGCGTGGACCA-3′. GAPDH was used for normalization of gene expression levels (reference Mm99999915-g1).

Mice immunizations were performed orally with sheep red blood cells for 2 wk and intraperitoneally with 10 µg LPS for 3 d. CD19 magnetic cell-sorted splenic B cells were used for AID-Chip experiments as previously described (Jeevan-Raj et al., 2011). For S_μ Q-PCR, data represent results obtained using two different primer pairs. S_μ forward 1 primer, 5′-TAAAATGCGCTAAACTGAGGTGATTACT-3′; S_μ reverse 1 primer, 5′-CATCTCAGCTCAGAACAGTCCAGTG-3′ (Kuang et al., 2009); S_μ forward 2 primer, 5′-TAGTAAGCGAGGCTCTAAAAAGCAT-3′; and S_μ reverse 2 primer, 5′-AGAACAGTCCAGTGTAGGCAGTAGA-3′ (Pavri et al., 2010). These primers were used with QuantiTect SYBR Green mix from QIAGEN. For J_H4 experiments, two primers (UPL 3′J_H4 forward, 5′-AGGGACTTTGGAGGCTCATT-3′; and UPL 3′J_H4 reverse, 5′-CTCCAACTACAGCCCCAACT-3′) have been designed and were used in combination with UPL probe#19 and Roche LightCycler 480 Probes Master mix. Splenic B cells were purified by CD43 magnetic cell sorting from nonimmunized mice and cultured in vitro with 10 µg/ml LPS for 3 d and used for Pol II–ChIP experiments as previously described (Tinguely et al., 2012). PCR primers were those used for analysis of IgH primary transcripts (probe located in the intron between the last J_H and the intronic E_μ enhancer; see sequences above).

Fig. S1 shows S_μ sequence and location of primers for S_μ PCR experiments.

We thank S. Desforges and B. Remerand for help with animal care.

This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC; SL 220100601332), Conseil Régional du Limousin, and Agence Nationale de la Recherche (Projets Blanc 2011). C. Vincent-Fabert was supported by a grant from the ARC.

The authors declare no competing financial interests.