Elucidation of IgH intronic enhancer functions via germ-line deletion

Abstract

Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iEμ) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iEμ (cEμ) were either deleted (cEμ^Δ/Δ mice) or replaced with a pgk-Neo^R cassette (cEμ^N/N mice). The cEμ^Δ/Δ mice had reduced B cell numbers, in association with impaired D to J_H and V_H to DJ_H rearrangement, whereas cEμ^N/N mice had a complete block in IgH V(D)J_H recombination, confirming that additional cis elements cooperate with iEμ to enforce D to J_H recombination. In addition, developing cEμ^Δ/Δ and cEμ^N/N B lineage cells had correspondingly decreased levels of germ-line transcripts from the J_H region of the IgH locus (μ0 and Iμ transcripts); although both had normal levels of germ-line V_H transcripts, suggesting that cEμ may influence IgH locus V(D)J recombination by influencing accessibility of J_H proximal regions of the locus. Consistent with chimera studies, peripheral cEμ^Δ/Δ B cells had normal surface Ig and relatively normal class switch recombination. However, cEμ^Δ/Δ B cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cEμ is not required for this process. The availability of mice with the iEμ mutation in their germ line will facilitate future studies to elucidate the roles of iEμ in V_H(D)J_H recombination in the context of IgH chromatin structure and germ-line transcription.

The mouse Ig heavy chain (IgH) locus can be divided into two principal regions: an ≈2.2-Mb 5′ region containing clusters of variable (V_H), diversity (D_H) and joining (J_H) segments and an ≈200-kb region downstream region harboring the sets of constant region exons (Cμ, Cδ, Cγ3, Cγ1, Cγ2b, Cγ2a, Cε, and Cα) referred as C_H genes. Generation of an Ig μ heavy chain protein, which is first expressed in B cell development, involves assembly of a V_HDJ_H exon upstream of Cμ via the V(D)J recombination reaction (1). In antigen-stimulated peripheral B cells, expression of downstream IgH isotypes (such as IgG, IgE, or IgA) can occur via a class switch recombination (CSR) reaction that replaces Cμ with a downstream set of C_H exons (2). Somatic hypermutation (SHM) of the variable region exon, which allows selection of higher affinity antibodies, also occurs in antigen-stimulated B cells (3). V(D)J recombination is initiated by the RAG endonuclease, whereas CSR and SHM are initiated by activation induced deaminase (4). The overall mechanisms that control these processes in the context of B cell development and activation are still being elucidated; however, many studies have implicated a role for transcriptional control elements (5). Major known control elements in the IgH locus include the transcriptional enhancer that lies within the intron between J_H and Cμ (6), which is referred to as the intronic IgH enhancer (iEμ), and the set of four enhancers that lie in the 3′ end of the IgH locus are referred to as the 3′ IgH regulatory region (RR) (7). Germ-line promoters flank V_H segments, D segments, and the sequences (S regions) that mediate CSR (2, 6).

Depending on the mouse strain, there are several hundred or more V_H segments embedded over several megabases at the 5′ end of the locus, followed by 13 D_H segments lying in the 100-kb region just 3′ of the V_H segments and 4 J_H segments, which lie just downstream of the D segments (6). The rearranged V_HDJ_H exon that encodes the variable region of IgH chains is assembled in developing B lineage cells via an ordered process. At the pro-B cell stage, D_H to J_H recombination occurs on both IgH alleles, followed by V_H to DJ_H joining. A productive V_HDJ_H rearrangement leads to the generation of a μ IgH chain that signals cessation of further V_H to DJ_H joining to effect allelic exclusion and development to the pre-B stage at which immunoglobulin light chain (IgL) gene rearrangement occurs (8). Expression of complete IgH/IgL Ig receptor leads to the differentiation of B cells that migrate to the periphery and can be stimulated to undergo CSR and SHM.

Control of V(D)J recombination, in the context of ordered rearrangement and feedback regulation, involves modulating differential accessibility of substrate V, D, and J segments to the RAG endonuclease (1). In this context, accessibility correlates with transcriptional activity of unrearranged (“germ line”) V_H, D, and J_H segments. Before D to J_H recombination, germ-line transcription is initiated at a promoter associated with iEμ/Iμ to generate Iμ transcripts and a promoter upstream of the DQ52 segment to generate μ0 transcripts (9, 10). Likewise, germ-line V_H genes are transcribed in the sense direction from V_H promoters before onset of V_H to DJ_H rearrangement, and such expression is down-regulated upon expression of a productive μ chain (11). More recently, abundant antisense transcripts of both genic and intergenic V_H regions have been described in ref. 12. Although the precise role of germ-line transcription remains unclear, transgenic recombination substrate studies showed that iEμ was necessary and sufficient to activate V(D)J recombination (13), and activation of germ-line promoters leads to chromatin structure changes that confer accessibility (14). However, studies of B cells from chimeric mice generated from ES cells that harbored mutations in which iEμ was deleted showed that deletion of iEμ impaired but did not totally block V(D)J recombination at the J_H locus, whereas pgk-Neo^R replacement of iEμ eliminated such rearrangements, implying redundant IgH cis-acting elements (15-17).

In addition to being initiated by activation-induced deaminase, both CSR and SHM require transcription through target sequences (S regions and variable region exons) (4). The activity of enhancer elements within the 3′ IgH RR (hs3b and hs4) is required to activate transcription from promoters flanking S regions (I promoters) of downstream C_H genes and, thereby, regulates CSR (2, 18). Based on location, iEμ was a candidate to transcriptionally activate Sμ during CSR; correspondingly, deletion of this element appeared to result in a decrease in IgH CSR (19, 20). However, the exact mechanism by which deletion of iEμ affects CSR remains unclear. The region of the IgH and Igκ light chain loci targeted for SHM encompasses a 2-kb stretch of DNA that includes the rearranged V_HDJ_H segment (21-23) with the 5′ boundary determined by the V_H or Vκ promoter (24-26). The Igκ locus also contains an intronic enhancer (iEκ) and a 3′ enhancer (3′Eκ) (27, 28). Transgenic studies suggested that both iEκ and 3′Eκ enhancers were required for SHM (29, 30). However, gene targeted mutation studies showed that the 3′Eκ is not required absolutely for SHM, implicating a larger or redundant role for the iEκ (31). Similarly, transgenic studies suggested a role for 3′IgH RR enhancers (hs3b and hs4) for SHM of the IgH locus (32), but gene-targeted mutation showed them to be dispensable (33). However, the role of iEμ in SHM of the endogenous IgH locus has never been tested.

To directly examine the role of iEμ in mediating IgH locus V_HDJ_H recombinational accessibility, effects on CSR, and potential roles in SHM, we have now generated and analyzed mice in which the cEμ is deleted in the germ line.

Materials and Methods

Gene Targeting. The targeting vector for the IgH Eμ core enhancer element was used to transfect 129 strain TC1 ES cells as described in ref. 17. Two ES cell clones showing homologous replacement of cEμ were injected into C57BL/6 blastocysts, chimeras harboring germ-line mutations mated to 129sv-ev mice to introduce the cEμ replacement mutation (“N” allele) into a pure 129 background. The replacement mutant mice were bred to EIIa-cre transgenic mice (34) to generate progeny that deleted the Neo^R cassette by cre-mediated recombination between loxP sites (generating the “Δ” allele).

Flow Cytometric Analysis. Single-cell suspensions were prepared, washed, and stained in PBS 2% FCS. For analysis staining, 0.5 × 10⁶ cells were incubated at 4°C for 45 min with various monoclonal antibodies conjugated to: phycoerythrin-CyChrome (anti-mouse B220 clone RA3-6B2, eBioscience, San Diego), phycoerythrin (anti-mouse CD43 clone S7 and anti-mouse CD4 clone H129.19, BD Biosciences Pharmingen, San Diego) and fluorescein-isothiocyanate (anti-mouse IgMa clone DS1 and anti-mouse CD8a clone 53-6.7, BD Biosciences Pharmingen). Cells were analyzed on a FACSCalibur apparatus (BD Biosciences Pharmingen).

Somatic Hypermutation Assays. Peyer's patch cells from wt and cEμ^Δ/Δ mice were stained in 1.5 ml of PBS/2% FCS with rat monoclonal anti-mouse B220 (clone RA3-6B2) conjugated with phycoerythrin-Cy-Chrome (eBioscience); peanut agglutinin (PNA) conjugated to fluorescein-isothiocyanate (Vector Laboratories, Burlingame, CA) and hamster anti-mouse FAS (clone Jo2) conjugated to phycoerythrin (BD Biosciences Pharmingen); and germinal center (GC) B cells (B220⁺ FAS⁺ PNA^hi) were sorted and collected on a FACSVantage (BD Biosciences), genomic DNA was extracted and the V_HJ558 to J_H4 intronic region was amplified by using high-fidelity Pfu polymerase (BD Biosciences Stratagene, San Diego), the 700-bp products were cloned into the pCR4Blunt-TOPO vector (Invitrogen, Paisley, U.K.), and sequences were analyzed via lasergene seqman ii software (DNAstar, Madison, WI).

ELISA Assays for CSR. Sera from 10-week-old cEμ^N/N, cEμ^Δ/Δ, and wt mice were analyzed for the presence of different IgH classes and subclasses by ELISA (18). Each ELISA titration was performed twice.

Assays for V_HDJ_H and DJ_H Rearrangements. For Eμ^Δ/Δ animals, bone marrow cells were isolated and B cells purified by positive magnetic sort on LS columns-MidiMacs (magnetic cell sorting, Miltenyi Biotec, Bergisch Gladbach, Germany) after labeling with anti-mouse CD19 coupled magnetic separation microbeads. The positive fraction was stained with rat monoclonal antibody anti-mouse B220 (clone RA3-6B2) conjugated with phycoerythrin-CyChrome (eBioscience), rat monoclonal anti-mouse CD43 (clone S7) conjugated to phycoerythrin, and mouse monoclonal anti-mouse IgMa (clone DS1) conjugated to fluorescein-isothiocyanate (BD Biosciences Pharmingen). The pro-B (IgM^- B220⁺CD43^hi) and pre-B (IgM^- B220⁺CD43^low) cell fractions were sorted and collected on a FACSVantage apparatus (BD Biosciences) and genomic DNA extracted. For Eμ^N/N animals, cells from total bone marrow were isolated and genomic DNA extracted. Genomic DNA was diluted and PCR performed to detect D to J_H and V_H to DJ_H rearrangements (35). Normalization of DNA amount in each diluted sample was estimated by PCR amplification of the IgH locus distal regulatory element hs4 by using HS4-5′ and HS4-3′ primers.

RT-PCR Analysis. Eμ^N/N, Eμ^Δ/Δ, and wt mice were backcrossed into a Rag2^-/- background to obtain double mutant animals. Bone marrow cells were isolated and the CD19⁺ pro-B cell fraction was purified by magnetic separation as described above. RNA preparation and reverse transcription was performed as described in ref. 12. For quantitative real-time PCR an iCycler (Bio-Rad) was used.

Hybridoma Analysis. Total splenocytes were stimulated with LPS or IL4/αCD40 in complete RPMI medium 1640 for 4 days and fused to NS-1 plasmacytoma cells. Hybridomas were generated, assessed for clonality, and then analyzed by ELISA for IgH isotype expression in the supernatant and VDJ_H recombination status as described in ref. 35.

Primers. All primers are listed in Table 1, which is published as supporting information on the PNAS web site.

Results

Germ-Line Replacement and Deletion of cEμ. To introduce germline cEμ mutations, we used a vector described in ref. 17 that replaces a 220-bp HinfI restriction fragment containing cEμ with a pgk-Neo^R gene flanked by loxP sites (Fig. 6, which is published as supporting information on the PNAS web site). TC1/129 ES cells were targeted, and two independent ES cell lines were identified in which the “loxP-Neo^R-loxP” cassette correctly replaced cEμ on one IgH allele (cEμ^N allele). Those clones were injected into C57/BL6 blastocysts and implanted into foster mothers to derive somatic chimeras. Chimeras were bred to either 129sv animals to obtain cEμ^N/+ heterozygous mutant animals or with EIIa-cre transgenic 129sv mice (34) to obtain animals in which the Neo^R gene was deleted via loxP/Cre-mediated recombination to obtain cEμ^Δ/+ mice, which are heterozygous for the “cleanly” deleted cEμ allele. The heterozygous mutant lines were bred, respectively, to obtain cEμ^N/N and cEμ^Δ/Δ animals.

Effects of cEμ^N/N and cEμ^Δ/Δ Mutations on B Cell Development. Previous analyses of cEμ function by targeted mutation used chimeric mice, which can be difficult to assess for subtle developmental defects because of the presence of host B lineage cells. Therefore, we compared B cell development in 8-wk-old germline mutant mice to that of wt counterparts (Fig. 1 A-D). Eμ^N/N animals had small spleens with only 30% of the absolute number of splenocytes compared to the wild type and lacked identifiable Peyer's patches and any detectable peripheral B cells (Fig. 1C and data not shown). In addition, Eμ^N/N mice had an essentially complete arrest of B cell development in the bone marrow at the B220^int CD43⁺ and/or B220^int cKit⁺ ProB1 stage where IgH locus V(D)J recombination is initatiated (Fig. 1 A and B). At 8 wk of age, all cEμ^Δ/Δ animals also exhibited smaller spleens with ≈50% the splenocytes of wt along with a reduced number of mature IgM⁺ B cells (Fig. 1C). However, the cEμ^Δ/Δ mice did show normal Peyer's patches (data not shown). Mature cEμ^Δ/Δ peripheral B cells showed no reduction in surface IgM expression or IgD (Fig. 1E and data not shown). In the bone marrow, there was a more modest impairment of B cell development as compared with the cEμ^N/N blockade, which was evidenced by a significantly increased percentage of B220^int CD43⁺ pro-B cells and a lower percentage of B220⁺ CD43^int pre-B cells (Fig. 1 A), again consistent with impairment of V(D)J recombination at the IgH locus. Peripheral and thymic T cells were normal in number and phenotype in both mutant lines (Fig. 1D and data not shown).

Fig. 1.

Flow cytometric analysis of B cell lineage populations. (A) FACS analysis of bone marrow cells from tibias and femurs of 129 wt, cEμ^N/N, and cEμ^Δ/Δ animals. Cells were stained with CyC anti-B220, FITC anti-IgMa, and PE anti-CD43 antibodies and gated on the IgM^- population. B220^int CD43⁺ pro-B and B220⁺CD43^lo pre-B cell populations are shown. (B) FACS analysis of bone marrow stained with CyC anti-B220 and FITC anti-CD117 (cKit) antibodies: B220^int cKit⁺ pro-B I population is shown. (C) FACS analysis of spleen cells stained with CyC anti-B220, FITC anti-IgMa antibodies: B220⁺ IgM⁺ mature B cell population is shown. (D) FACS analysis of spleen cells stained with CyC anti-CD3, FITC anti-CD4, and PE anti-CD8 antibodies: CD4⁺ and CD8⁺ mature T cell populations are shown. (E) Comparative IgM surface expression of B220⁺ splenocytes from cEμ^Δ/Δ and 129 wt animals. Mice were analyzed at 4-6 wk of age.

Impaired D_H to J_H and V_H to DJ_H Rearrangement in cEμ^N/N and cEμ^Δ/Δ Mice. To confirm that the cEμ replacement with a pgk-Neo^R cassette blocked V(D)J recombination at the IgH locus, we used a PCR-based approach to assay for D to J_H and V_H to DJ_H rearrangements in DNA from total cEμ^N/N bone marrow and found these rearrangements to be essentially absent (Fig. 7, which is published as supporting information on the PNAS web site), demonstrating that the absence of peripheral B cells in these mice corresponds to an essentially complete block of IgH locus V(D)J recombination. A similar PCR analysis of IgH locus V(D)J recombination performed on DNA isolated from sorted cEμ^Δ/Δ pro- and pre-B cells revealed that D-J_H rearrangements were not markedly impaired as detected by this assay (Fig. 2 Top and Top Middle; also see below). In contrast, cEμ^Δ/Δ pro- and pre-B cells had a substantial decrease in V_H to DJ_H rearrangements (Fig. 2 Top Middle, Bottom Middle, and Bottom). Together, these latter findings support the suggestion that deletion of iEμ has a more major affect on V_H to DJ_H than on D to J_H rearrangement (17).

Fig. 2.

IgH D-J_H and V_H-DJ_H rearrangement assays. Genomic DNA from sorted pro-B and pre-B cell populations from 129 wt and cEμ^Δ/Δ animals was subjected to PCR amplification of D_H to J_H and V_H to DJ_H rearrangements. PCR reactions were performed on serial 3-fold dilutions and products detected via Southern blot hybridization by using an oligonucleotide probe specific to J_H4. Kidney DNA was a negative control. Another independent PCR assay amplifying the 3′ IgH regulatory hs4 enhancer was performed to normalize DNA input. Mice were analyzed at 6 wk of age.

To assay more sensitively for potential defects in D to J_H and V_H to DJ_H rearrangements in cEμ^Δ/Δ B lineage cells at a clonal level, we generated splenic B cell hybridomas from wt and cEμ^Δ/Δ splenic B cells and analyzed the rearrangement status of their J_H alleles by Southern blotting. Normal B cells generate D to J_H rearrangements on both J_H alleles. Correspondingly, we found that only 5% of wt B cell hybrdomas contained a germ-line J_H allele (Fig. 3A), consistent with other studies (17, 35). The occurrence of a small percentage of hybridomas with germ-line alleles is thought to reflect a low level of tripartite fusions with nonlymphoid cells (35). In contrast, ≈30% of cEμ^Δ/Δ hybridomas retained the J_H locus in germ-line configuration on one allele, consistent with defect in D to J_H rearrangement efficiency (Fig. 3A) that is below the level readily observed by the PCR assay. These hybridoma results that indicated defective D to J_H rearrangement in the cEμ^Δ/Δ background were confirmed by Southern blotting analysis of LPS-stimulated splenocyte DNA that showed that activated B cells from cEμ^Δ/Δ animals, but not wt animals, retained a detectable level of the 6.2-kb germ-line J_H-hybridizing EcoRI fragment (Fig. 3B). All of the hybridomas must have a productive V_HDJ_H rearrangement. Therefore, to assay for V_H to DJ_H recombination defects, we asked what portion of hybridomas that had rearranged both J_H alleles had V_HDJ_H versus a DJ_H rearrangement on their nonproductive allele. Normally, ≈50-60% of wt B cell hybridomas have the nonproductive J_H allele in the DJ_H configuration, and 40-50% have the nonproductive allele in the V_HDJ_H configuration. However, we found that 88% (87 of 99) of cEμ^Δ/Δ hybridomas had the nonproductive allele in the DJ_H configuration (Fig. 3A), confirming the V_H to DJ_H joining defect indicated by the PCR-based studies with pro-B cells (Figs. 2 and 7).

Fig. 3.

IgH rearrangement status of 129 wt and cEμ^Δ/Δ peripheral B cells. (A) V_HDJ_H rearrangement status of hybridomas obtained after fusion of splenic wt and cEμ^Δ/Δ B cells. Rearrangement status was assayed by Southern blotting (35). Results of three independent fusions are shown. (B) Rearrangement status of nonproductive IgH alleles in peripheral B cells. DNA prepared from positively sorted splenic B cells was digested with EcoRI and the 6.2-kb restriction fragment corresponding to the germ-line configuration was detected by Southern blotting with a 0.47-kb HindIII-NaeI fragment containing the J_H4 segment. On the same blot, DNA normalization was controlled by hybridization to a 0.8-kb EcoRI-StuI fragment specific of the ≈22 kb nonrearranged 3′IgH RR EcoR1 fragment.

Role of cEμ in Germ-Line IgH Transcription. It was suggested previously that the iEμ regulates μ0 transcripts, which originate 5′ of DQ52 (10), and Iμ transcripts, which originate immediately 3′ of the Eμ core (9). To look at expression levels of these transcripts, Eμ^N/N and Eμ^Δ/Δ mice were crossed into a Rag2-deficient background. Bone marrow cells were magnetic-activated cell sorted for CD19 surface expression, which allowed us to generate an ≈90% pure pro-B cell population. RT-PCR analysis of Eμ^Δ/Δ pro-B cells revealed a 10- to 20-fold decrease of Iμ and μ0 transcript levels compared with those of wt mice; in these experiments, the level of λ5 (Fig. 4A) and Rag1 (Fig. 4B) transcripts, respectively, were used to standardize RNA levels. Eμ^N/N pro-B cells also exhibited an approximately 10- to 20-fold decrease in Iμ transcripts and, strikingly, μ0 transcripts were not detectable in these cells by either semiquantitative (Fig. 4A) or quantitative (Fig. 4B) RT-PCR. In contrast to our findings for Iμ and μ0 transcripts, we found that germ-line transcript levels from various Eμ proximal and distal V_H families were similar in cEμ^N/N, cEμ^Δ/Δ, and wt mice (Fig. 4A).

Fig. 4.

IgH locus germ-line transcripts in wt and cEμ mutants B lineage cells. (A) Pro-B cell cDNA from bone marrow of Rag2-deficient wt, cEμ^N/N, and cEμ^Δ/Δ mice was subjected to PCR analysis to detect the indicated transcripts, serial dilutions were 10-fold, and λ5 transcripts were used as an input control. (B) Quantitative PCR analysis of pro-B cell cDNA from bone marrow of Rag2-deficient wt (lane 1), cEμ^N/N (lane 2), and cEμ^Δ/Δ (lane 3) mice with primers amplifying μ0 transcripts. Transcript levels were standardized to Rag1 expression.

Reduced Class Switch Recombination in the Absence of the cEμ. To investigate IgH class switching in vivo in the absence of the cEμ intronic enhancer, we stimulated total splenocytes from wt and Eμ^Δ/Δ mice, respectively, with LPS (which induces CSR to IgG2b and IgG3) or with αCD40 plus IL-4 (which induces CSR to IgG1) and produced clonal hybridoma lines that were screened for IgH isotype production by ELISA. Analyses of the hybridomas showed that IgH class switching, under either condition, was, at most, modestly diminished (Fig. 8A, which is published as supporting information on the PNAS web site). In this regard, we also quantified, by ELISA, each IgH subclass in the serum of 10-wk-old mutant animals. A cohort of nine cEμ^Δ/Δ animals compared with six wt littermates, again revealed, at most, a modest decrease in some, but not all, downstream IgH isotypes (Fig. 8B). Although the defects observed by the above assays were only of borderline significance, it is known that subtle defects in class-switching at the cellular level can become more obvious when CSR is assayed on the second allele of hybridomas that had undergone CSR on one allele (36). In this context, normal B cells undergo CSR on both IgH alleles at very high frequency (>90%); however, of 29 cEμ^Δ/Δ hybridomas analyzed, only 16 (55%) had undergone CSR on the second allele, clearly documenting a role for cEμ in maintaining CSR at its highest efficiency, consistent with earlier findings based on analyses of chimeric mice (19).

Normal Somatic Hypermutation in cEμ^Δ/Δ B Cells. Because the cEμ^Δ/Δ mutation allows B cell development to proceed at considerable levels, we were able to assay for the effects of this mutation on SHM of IgH genes. In two independent experiments, comparing three cEμ^Δ/Δ animals and two wt littermates, we PCR amplified and cloned IgH variable region exons and downstream flanking sequences from sorted Peyer's patch GC B cells. We quantified mutations in the 500-bp region just downstream of the J_H4 exon and found the mutation frequency in cEμ^Δ/Δ GC B cells to be comparable with that observed in wt controls (Fig. 5A). Thus, when comparing the number of mutations per sequence, we found that cEμ^Δ/Δ GC B cells displayed an approximately normal fraction of sequences that were highly mutated (i.e., with 10 or more mutations in the 500-bp region analyzed: 29% compared with 38% in wt; Fig. 5B). On the other hand, cEμ^Δ/Δ B cells had a 2-fold increase of unmutated sequences (31% in mutants and 14% in wt, Fig. 5B), which might reflect fewer mutant cells entering the GC reaction because of the decreased numbers of peripheral cEμ^Δ/Δ B cells. Finally, we analyzed the pattern of nucleotide substitutions in the absence of the cEμ and found that transitions and transversions occurred about the same frequency at dC/dG or dA/dT (Fig. 5C), suggesting that cEμ is not involved in targeting of particular types of mutations.

Fig. 5.

Somatic hypermutation of the IgH locus in 129 wt and cEμ^Δ/Δ mice. GC B cell DNA was isolated from B220⁺ PNA^hi Fas⁺ Peyer's patches cells of a nonimmunized 10-wk-old animal. The IgH variable region was amplified as described in ref. 37. (A) Number of mutations versus total length of DNA sequence analyzed, and the mutation frequency in two wt and three mutant animals. Only sequences that contain at least one mutation were included, and clonally related sequences were excluded. (B) Pie plots showing the number of mutations (at the perimeter of the plot) and the percentage of the total number of clones for each percentage group (proportional to the area in each slice); the total number of clones analyzed for each genotype is noted in the center. (C) Pattern of nucleotide substitutions detected in the J_H4 intronic region. The table shows the percent of mutations for each type of nucleotide present in the germ-line sequence (vertical axis) to a different nucleotide (horizontal axis). The table below compares the ratio of mutations at dC/dG to those at dA/dT as well as the ratio of transitions: transversions substitutions at both dC/dG and dA/dT.

Discussion

There have been several previous mutational analyses of iEμ function done by an approach in which the targeted Eμ replacements were generated in ES cells, which then were used to analyze the effects of the mutations in the context of mutant B cells generated in chimeric mice (15-17, 19). This approach demonstrated that deletion of cEμ leads to decreased V(D)J recombination at the IgH locus, with the most predominant effect being on V_H to DJ_H rearrangement (15-17), and also demonstrated that cEμ influences IgH class switch recombination (19). Now, we have generated a line of mice that harbor a deletion of cEμ in their germ line. Analyses of these mice confirmed and extended a number of findings made with chimeric mice, including a more detailed analysis of the requirement for iEμ for normal B cell development and confirmation of the existence of an additional element that influences D to J_H rearrangement. In addition, our current analyses have provided previously undescribed insights into iEμ function. Thus, we show that the influence of cEμ on V(D)J recombination at the J_H locus correlates well with its effects on germ-line transcription through this region. In addition, we have made the unexpected finding that cEμ is not required for SHM of IgH variable region exons.

It was previously shown that deletion of iEμ substantially impairs V_H to DJ_H rearrangement at the IgH locus. We have confirmed this conclusion and clearly documented a significant inhibition of D to J_H rearrangement as well. It has been speculated that promoter activity associated with the generation of μ0 transcripts was required for efficient D-J_H transcription. The dramatic decrease in μ0 levels observed in cEμ^Δ/Δ pro-B cells suggests that cEμ directly regulates the μ0 promoter and strengthens the hypothesis that this interaction is linked to V(D)J recombinational accessibility of the J_H locus. Moreover, our finding that sense germ-line V_H transcription appears relatively normal in cEμ^Δ/Δ pro-B raises the possibility that the dramatic influence of cEμ on V_H to DJ_H rearrangement may be mediated via the activity of this element with respect to the D-J_H versus the V_H portion of the IgH locus. For example, the cEμ may be required to activate DJ_H rearrangement for recombination with upstream V_H segments (6). However, another possibility would be an effect on anti-sense V_H transcription, which can now be analyzed in the context of the homozygous germ-line iEμ mutation on a RAG-2 deficient background.

In the absence of cEμ, we still observed the development of substantial levels of peripheral B cells, in accord with the observation that cEμ deletion diminishes, but does not block, IgH locus V(D)J recombination. In contrast, replacement of iEμ with a pgk-Neo^R gene totally blocked B cell development at the pro-B stage and, correspondingly, completely blocked D to J_H rearrangement and assembly of variable region exons (refs. 15-17 and this study). Although the precise mechanism by which replacement of iEμ with a pgk-Neo^R gene fully blocks V(D)J recombination remains speculative, we now demonstrate that this mutation, in contrast to the iEμ deletion, fully abrogates expression of μ0 transcripts, most likely due to the effect on transcriptional initiation. Thus, the correlation between the effects on transcription and V(D)J recombination of these two mutations are strongly suggestive of a cause/effect relationship. In this context, studies with V(D)J recombination substrates have shown that levels of germ-line transcription correlate precisely with V(D)J recombination efficiency (38) and that promoters have an important role in V(D)J recombinational accessibility (14). There are several mechanisms by which the pgk-Neo^R replacement might inhibit transcription from the μ0 promoter (and D to J_H rearrangements). One possibility is that the cassette may block μ0 promoter activation by inhibiting an unknown cis-regulatory element via a promoter competition/insulating mechanism. In this regard, elements in the 3′ IgH RR would be attractive candidates because they work over at least 200-kb (39), and their activity has been shown to be inhibited by insertion of a pgk-Neo^R cassette between them and their target I region promoters (18, 39-41). Alternatively, pgk-Neo^R might alter local chromatin structure and, thereby, interfere with germ-line transcription and D to J_H recombination. The availability of RAG-2 deficient mice harboring the iEμ mutations in their germ line will allow the latter possibility to be addressed directly.

It has been shown previously in chimeric mice that deletion of the cEμ somewhat diminishes but does not abrogate CSR (19, 20). Likewise, we have observed only a very modest defect in IgH class switching in cEμ^Δ/Δ B cells that was hardly apparent with respect to serum IgH isotype levels but was demonstrable at the level of CSR on the nonexpressed allele in hybridomas. We could further demonstrate that the iEμ deletion led to a marked decrease in the level of Iμ transcripts, although any direct relationship remains to be determined. We also found normal surface Ig expression on peripheral cEμ^Δ/Δ B cells, suggesting that cEμ may not be essential for normal expression of rearranged V(D)J segments; although it will be necessary to look at transcription levels to fully address this issue. In this regard, it appears that the two distal IgH 3′ elements (hs3b and hs4) have a more important role (18), although redundant activities between iEμ and 3′ IgH regulatory region elements or other unknown elements cannot be excluded. These findings are reminiscent of findings that deletion of the intronic Igκ light chain locus enhancer also does not dramatically affect expression of this locus (42), whereas deletion of the 3′Eκ does (43). Finally, we have shown that cEμ is dispensable for substantial levels of SHM of rearranged IgH variable region exons. In this context, our findings also imply, perhaps surprisingly given findings from transgenic studies (23, 44, 45), that cEμ is not necessary to target the SHM machinery to IgH variable region exons, although additional studies will be necessary to determine whether there is any alteration in the overall DNA sequences targeted. In any case, these results indicate that other elements must function, at least in part, to fulfill the SHM targeting function at the IgH locus.