J. Exp. Med.
© The Rockefeller University Press
0022-1007/97/01/239/12 $2.00
Volume 185 January 1997 239-250

The Transcriptional Promoter Regulates Hypermutation of the Antibody Heavy Chain Locus

By Kathleen Tumas-Brundage and Tim Manser

From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, Pennsylvania 19107

Summary

Materials and Methods

Results

Discussion

Footnotes

Acknowledgements

References

Summary

Antibody variable (V)¹ gene hypermutation results in diversification of the antibody repertoire in a variety of vertebrates (1). In mice and humans, this process is induced during T cell-dependent immune responses, is intimately associated with differentiation of memory B cells, and in combination with antigen affinity based selection, results in affinity maturation of serum antibodies (2). Past retrospective and descriptive studies in mice have shown that hypermutation introduces mainly single, untemplated nucleotide replacements at a rate estimated to be 10³/bp/cell division (3), acts efficiently only in and immediately around fully rearranged L chain V and V_H genes (i.e., in a "zone" from ~300-bp 5 to 1-kb 3 of the V gene) (7), and is not mechanistically linked to isotype switch or V(D)J recombination (13).

Current data lend the most support to models for the mechanism of hypermutation that invoke a role for the transcription apparatus. Mutations are rare in regions 5 of V gene promoters, suggesting a "boundary" (8, 11, 12, 17). The distribution of mutations in and around a V gene appears to be influenced by the location of regions flanking the 5 side of the V coding sequence and not the V coding sequence itself (10, 17). Since 5 regions distal to V coding sequence are clearly not required for hypermutation (18, 19), there may be an influence of the promoter region on distribution. More recent studies using transgenic technology have begun to shed light on the cis-acting elements necessary for hypermutation of V L chain genes. A role for both the intronic and 3 distal  transcriptional enhancers in influencing the rate of hypermutation of the Ig locus has been implicated (19), and the insertion of a V promoter just 5 of the C exon in an Ig transgene results in somatic mutation of this normally unmutated region (20). While it is not known whether transcription is required for hypermutation or whether the influence of promoters and enhancers on hypermutation is secondary to their role as transcriptional regulators, these data nevertheless suggest that common cis-acting elements are involved in these processes.

Materials and Methods

Transgenic Mice. Transgenic mice were generated by standard methods (21) using C57BL/6 × C3H F1 mice and linearized plasmid constructs. Founder mice were backcrossed for two ("AA" mice), three ("3A" mice), or four ("4A" mice) generations to the A/J strain before use. All mice used in these experiments were heterozygous for multicopy transgenic arrays. Mice were housed under specific pathogen-free conditions and given autoclaved food and water. All experimental procedures on mice were conducted according to the National Institutes of Health guidelines.

Generation and Screening of Hybridomas. All hybridomas were generated from the spleens of 8-10-wk-old transgenic mice immunized with the same preparation of p-azophenylarsonate (Ars)- KLH intraperitoneally using one of the following immunization protocols: 10 d after a single injection of 100 µg Ars-KLH in CFA (late primary), 3 d after secondary or tertiary boosting injections of 100 µg Ars-KLH in PBS at least 30 d after priming with 100 µg Ars-KLH in CFA, or 3 d after a regimen of priming with 100 µg Ars-KLH in CFA, a 7-d rest, and then four injections of 25-50 µg Ars-KLH in PBS spaced at 2-d intervals (fusion 16 d after initial priming, hyperimmunized primary). Supernatants from the hybridomas were screened by ELISA for those expressing the transgenic V_H gene using the antiidiotypic antibodies E4 and 107 as previously described (15). E4 recognizes all canonical anti-Ars antibodies. 107 recognizes only canonical anti-Ars antibodies partially encoded by the 36-65 V_H gene, due to the rare V_H-D and D-J_H junctional amino acids encoded by this gene (22, 23).

Reverse Transcriptase-PCR, Genomic PCR, and Nucleotide Sequencing. Total RNA was prepared from hybridomas, reverse transcriptase-PCR performed, and PCR products directly sequenced as previously described (24). The hybrid loci were amplified from hybridoma genomic DNA by PCR, and bulk PCR products were directly sequenced also as described previously (18, 25). Since bulk PCR products were sequenced in all experiments, the contribution of Taq polymerase error to mutations in the final sequences was considered to be insignificant.

Results

If Ig promoters contain cis information necessary for the locus-specific action of hypermutation, replacement of the natural promoter in an Ig locus with a heterologous, but B cell-specific promoter should ablate hypermutation. Moreover, if the site specificity of mutation is regulated by the promoter, changing the location of this element relative to the VDJ coding sequence and downstream regulatory elements such as the intronic enhancer in an Ig locus should cause a corresponding change in the location of the characteristic "zone" of mutation. To test these predictions for the IgH locus and its promoter we created transgenic mice using two modified forms of a construct containing an antiArs VDJ gene called 36-65 from A/J mice, a minimal V_H promoter (145 bp containing only the cap/initiation site, a TATA box, an octamer, a heptamer, and a purine rich region), and 1.5 kb of natural 3 flanking sequence. In one construct, the natural V_H promoter was replaced by the heterologous, but B cell-specific minimal B29 (Ig-) gene promoter. In the second construct, the size of the intron between the V_H leader exon and the main V_H exon was enlarged 750 bp via insertion of a portion of a Drosophila melanogaster myosin heavy chain gene intron. Since all of the cis-acting elements necessary for hypermutation of V_H transgenes present at ectopic chromosomal sites have yet to be defined (18, 26), we exploited a novel somatic recombination pathway (18, 25) to introduce our modified V_H constructs into a natural context within the endogenous (i.e., nontransgenic) H chain locus (Fig. 1).

We have previously shown (18, 25) that transgenes derived from constructs containing the 36-65V_H gene and various amounts of natural 5 and 3 flanking DNA (hereafter referred to as wild type) recombine with the endogenous IgH locus at a low frequency in B cells, giving rise to fully functional "hybrid" H chain genes that undergo normal hypermutation (Fig. 1). In such hybrid loci, the VDJ and its 5 flanking sequences are transgene derived, and regions 3 of the J_H3-J_H4 region are derived from the endogenous H chain locus. The kinetics and distribution of hypermutation are not altered in such hybrid loci, and the generation of these loci is not mutagenic or mechanistically linked to the hypermutation process (18, 24, 25).

Founder mice were crossed with A/J mice to introduce a "canonical" V gene (V10J1), that normally is coexpressed with canonical V_H genes, encoded by the same V_H, D, and J_H gene segments as the 36-65V_H gene, in a major fraction of the B cells that respond to Ars immunization in the A/J mouse strain (13). One line of mice obtained from the B29 promoter construct, termed BP2, and two lines of mice created with the enlarged intron construct, termed CPM1 and CPM2, were immunized with Ars-KLH and hybridomas generated from late primary and secondary immune responses to sample the hypermutated B cell population. Hybridomas coexpressing H chains encoded by a hybrid locus and canonical endogenous L chains were identified using an antiidiotypic antibody specific for the 36-65 V region (18, 24). The presence of hybrid loci in hybridoma genomic DNA was confirmed by Southern blot analysis (data not shown).

Sequencing of the V_H genes present in B29 promoterdriven hybrid loci expressed by late primary immune response hybridomas via reverse transcriptase-PCR revealed that these genes contained few or no somatic mutations. Analysis of V_H genes in such hybrid loci expressed by secondary and tertiary hybridomas showed that they were mutated, but also at levels lower than expected (Table 1). However, the average frequency of mutation in expressed, canonical V (V10J1) genes in these hybridomas was comparable to that observed in canonical V genes expressed by control hybridomas (see legend to Table 1 and Materials and Methods for details). The low frequency of mutation observed in canonical V genes was anticipated since previous studies on Ars-induced hybridomas derived from A/J mice have shown that the frequency of canonical V mutation is two to threefold lower than of canonical V_H mutation (10, 15). Chi-squared comparison of the frequency of mutations in the V genes expressed by hybridomas derived from BP2 transgenic mice revealed the frequency difference between endogenous canonical V_H genes and B29 promoter-driven 36-65V_H genes present in hybrid loci was highly significant (P <10⁴). This difference in mutation frequency is particularly apparent among primary hybridomas. The significance of this difference is further supported by the fact that hybridomas expressing hybrid or endogenous H chain loci were often derived from the same mouse, or from littermates immunized with the same antigen preparation and from which fusions were performed at the same time (see legend to Table 1). In contrast, the mutation frequencies observed in the endogenous canonical V genes expressed by these two groups of hybridomas were not significantly different (P ~0.9). Thus, the hypermutation process had operated normally in trans in the precursors to the hybridomas expressing B29 promoter-driven hybrid loci.

Table 1. Somatic Hypermutation in BP2, CPM1, and CPM2 Hybridomas Name of hybridomas* Isotype Mutations in VH Mutations in V BP2 hybridomas expressing hybrid H chain loci   Primary     4ABP2-8 G7 IgG2b  0 1 D10 IgG2b  0 0 E4 IgG2b  0 1     4ABP2-16 G6 IgG1  1 3 A5 IgG1  2 0     AABP2-2 E3 IgG2b  2 3     3APB2-7 H3 IgG2b  0 2   Secondary and tertiary     AABP2-28a E5 IgG1  1 1     3ABP2-59 A12 IgG1  7 1 D7 IgG2b  2 1     4ABP2-13 H7 IgG1  4 0     3ABP2-44 H8 IgG1  1 2 BP2 hybridomas expressing endogenous H chain loci   Primary     4ABP2-8 E7 IgM  3 0     AABP2-10 H7 IgG1  5 1     3ABP2-7 B1 IgG2b  4 4     4ABP2-23 F10 IgM  1 3 D2 IgG1  3 1   Secondary and tertiary     3ABP2-43 D3 IgG1  8 1 A12 IgG1 11 2     3ABP2-45 G2 IgG1 3 2 CPM hybridomas expressing hybrid H chain loci   Primary     3ACPM2-23 B2 IgG1  3 1 G6 IgG1  5 3     4ACPM1-2 D9 IgG1  2 4 G3 IgG1  0 4 C6 IgG3  0 1     4ACPM1-3 D5 IgG2b  3 2 F1 IgG3 12 2 *  The first four-five characters before the dash in each hybridoma name indicate the name of the mouse from which that hybridoma was derived. Among the BP2 hybridomas, the groups (4ABP2-8 D10, E4, E7, G7); and (3ABP2-7 B1, H3) were derived from single mice. The groups (3APB2-44 H8, 3ABP2-45 G2); (AABP2-2 E3, AABP2-10 H7) and (4ABP2-16 G6, A5, 4ABP2-23 D3, A12) were derived from littermates immunized with the same dose and preparation of antigen and sacrificed for fusion on the same day.

Low levels of V_H mutation were also observed on average in enlarged leader intron hybrid loci expressed by late primary response hybridomas, and many such loci contained no V_H mutations. As was the case for the hybridomas containing B29 promoter-driven hybrid loci, the canonical V mutation frequency in hybridomas containing enlarged leader intron hybrid loci appeared normal (data from seven representative hybridomas are shown in Table 1). As summarized in Fig. 2, the mutation frequencies in the V_H genes in both types of hybrid loci are well below that previously determined for wild-type 36-65V_H hybrid loci at times late in the primary response.

The frequency of V_H gene mutation in hybrid loci is not directly reflective of their intrinsic rate of mutation, as many mutations in coding region can be selected in vivo. Fig. 3 shows the sequence of the expressed V_H genes in the B29 promoter-driven and enlarged leader intron hybrid loci that contain at least one somatic mutation. Of the eight V_H genes in B29 promoter-driven hybrid loci, six contain somatic mutations at positions 58 and 59 in CDR2 that are recurrently observed among canonical V_H genes. Two such mutations have been shown to result in increased affinity for Ars (27). In three such V_H genes, these recurrent mutations are the only mutations observed, and in one V_H gene, a recurrent mutation is one of only two mutations. Of these four V_H genes (those expressed by hybridomas 4ABP2-16G6, AABP2-28aE5, 3ABP2-59D7, and 3ABP2-44H8), three are expressed by hybridomas isolated from secondary or tertiary responses. In wild-type 36-65V_H genes in hybrid loci and endogenous canonical V_H genes, particularly those expressed by secondary and tertiary hybridomas, such mutations are usually accompanied by many other "selectively neutral" (e.g., silent) mutations (18, 28). Of the nine mutated enlarged leader intron hybrid loci, five contain recurrent mutations. These data indicate that the frequency of V_H coding region mutation is an overestimate of the reduced intrinsic mutation rate in this region in both types of modified hybrid loci, particularly those driven by the B29 promoter.

Two explanations were considered for these observations: (a) the intrinsic rate of hypermutation throughout the modified hybrid loci was reduced; and (b) the site specificity of hypermutation was altered such that the V_H gene no longer was present in the region of "peak" mutational activity. To distinguish between these possibilities, hybrid loci were cloned from hybridoma DNA and regions flanking the 5 and 3 sides of the V_H gene were sequenced. Analysis of five B29 promoter-driven hybrid loci chosen to represent the entire range of V_H mutation frequency (i.e., 0 -7 V_H mutations, hybridomas AABP2-2E3, 3ABP2-59A12, and 4ABP2-13H7, -16A5, and -8D10) revealed that the distribution of mutation was similar to wild-type 36-65V_H hybrid and endogenous canonical V_H loci. However, mutation frequencies in both 5 and 3 flanking regions were low. In the 300 bp of DNA flanking the 5 side of VDJ, only one mutation was observed in the 1.5 kb sequenced. In the 1.5 kb of DNA flanking the 3 side of VDJ, only 23 mutations were observed in the 7 kb sequenced. However, 15 of these mutations were observed in the first 300 bp 3 of the VDJ. In previous analyses of the distribution of mutation around endogenous canonical V_H genes and 36-65V_H genes present in wild-type hybrid loci, 12-14% of the total mutations were found 5 of the VDJ and 86-88% were found 3 of VDJ (17, 18). Moreover, the majority of 3 mutations were observed within the first 300 bp 3 of VDJ (see below). The chemical nature of mutations observed in B29 promoter-driven hybrid loci was also characteristic of bona fide hypermutation (29-32, and see below). Thus, the effect of replacing the natural V_H promoter with the B29 promoter appears to only be a reduction in the rate of hypermutation.

Sequencing of DNA flanking the 5 side of the V_H gene in seven enlarged leader intron hybrid loci revealed high frequencies of mutation in both the Drosophila intron and natural Ig intron regions (Fig. 4). However, few mutations were observed in the region flanking the 3 side of V_H in these hybrid loci, a region that, as discussed above, displays a high frequency of mutation in wild-type 36-65V_H hybrid and endogenous canonical V_H loci (Fig. 5). These data indicate that the distribution, but not the rate, of mutation was altered due to the lengthening of the leader intron.

Table 2. Location and Type of Somatic Mutations in Enlarged Leader Intron Hybrid Loci Nucleotide 3ACPM2-23 4ACPM1-2 4ACPM1-3 B2 G6 D9 G3 C6 D5 F1 Vector   1 3 of Promoter   113 GTC GGC   116 CTC CAC Leader intron   186 TAA TCA   202 GGT GCT   209 ATT AAT Dros. intron   247 TAG TTG   249 CA CCA   263 TGT TCT   276 start TGT TAT   306   CAT CGT   324   TAC TCC   367     369   CAC CTC   375   AAC AGC AAC AGC   391   GTA GAA   400   ATG AGG   414   GAA GCA   417   TAT TGT   427   AAG AGG   428   AGT AAT   431   TAT TTT   436   CTT CAT   441   AGT ACT   466   CA CCA   472   AGC AAC   479   GAT GGT   489   AAA ACA   509   AAT ATT   519   start   520   TAA TGA     531   TAA TTA     565   GAT GGT     576   AGT AAT     579   AAT ACT     583   TAC TGC     592   TAT TTT     595   GTA GCA     613   TAT TCT     634   TAC TTC     638   TAA TGA     646   TAC TCC   Nucleotide 3ACPM2-23 4ACPM1-2 4ACPM1-3 B2 G6 D9 G3 C6 D5 F1   701   TAT TTT     703   TAG TGG     705   GCA GTA     706 end     712 AGC AAC     715 AAT AGT     721 AGC ATC     726 TTC TGC     734 TTA TAA     735 TAT TTT