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Regulation of IgH Gene Assembly: Role of the Intronic Enhancer and 5'D_Q52 Region in Targeting D_HJ_H Recombination¹

Roshi Afshar^*, Steven Pierce^*, Daniel J. Bolland, Anne Corcoran and Eugene M. Oltz^2,*

^* Department of Microbiology/Immunology, Vanderbilt University Medical School, Nashville, TN 37232; and Laboratory of Chromatin and Gene Expression, The Babraham Institute, Babraham, Cambridge, United Kingdom

	Abstract

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The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Eµ) only modestly reduce D_HJ_H rearrangements when assessed in peripheral B cells. However, deletion of Eµ severely impairs recombination of V_H gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Eµ deletions on primary D_HJ_H rearrangement: 1) Accessibility at the D_HJ_H cluster is controlled by a redundant cis-element in the absence of Eµ. One candidate for this element lies 5' to D_Q52 (PD_Q52) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, D_HJ_H recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PD_Q52 and Eµ in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PD_Q52 promoter is dispensable for germline transcription and recombination of the D_HJ_H cluster. In contrast, we demonstrate that Eµ directly regulates accessibility of the D_HJ_H region. These findings reveal a significant role for Eµ in the control mechanisms that activate IgH gene assembly and suggest that impaired V_HD_HJ_H rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in D_HJ_H recombination.

	Introduction

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The enormous diversity of Ag receptors required for mammalian immunity is generated in precursor lymphocytes by a genetic process, termed V(D)J recombination. Each precursor lymphocyte assembles unique Ig or TCR genes via the random selection of one variable (V), one diversity (D), and one joining (J) gene segment from large arrays of these elements (1, 2, 3, 4). Each of these recombination events is mediated by a single enzyme complex that contains the lymphocyte-specific proteins RAG-1 and RAG-2 (5, 6). The RAG-1/2 complex binds to conserved DNA elements, termed recombination signal sequences (RSSs),³ which flank all V, D, and J segments (1, 7). Following the synapsis of two compatible RSSs, the RAG proteins introduce dsDNA breaks precisely at the RSS/coding region borders, resulting in a pair of coding and signal ends. Subsequently, the coding and signal ends are ligated by ubiquitous double-strand break repair machinery to produce chromosomal coding joins and signal joins, which are normally deleted as an extrachromosomal circle (1, 2, 8).

Although all Ag receptor loci use a common RAG complex and RSS substrates for their assembly, V(D)J recombination is regulated by stage-, allele- and tissue-specific mechanisms. For example, Ig genes rearrange efficiently in precursor B cells, whereas TCR genes remain frozen in their germline configuration (4). The process of Ig gene assembly is also highly ordered, initiating at the pro-B cell stage with recombination between 1 of 13 D_H gene segments and 1 of 4 J_H segments. Subsequent recombination between a preformed D_HJ_H element and 1 of 150 V_H gene segments completes the assembly of an Ig V region exon (9, 10, 11). Productive rearrangement of an IgH allele by a pro-B cell stimulates progression of that clone to the pre-B cell stage, blocks further V_HD_HJ_H recombination (allelic exclusion), and signals for initiation of IgL chain gene assembly (9, 10). In-frame rearrangement of an IgL chain gene (Ig or Ig) enables the precursor cell to express its signature BCR and to differentiate into a naive B cell clone (9).

The tissue- and stage-specific aspects of V(D)J recombination are largely controlled by changes in the accessibility of chromatin at targeted gene segments (12). Recombinationally active gene segments are generally transcribed, reflecting an open chromatin configuration that is accessible to both V(D)J recombinase and the transcriptional machinery (13, 14, 15, 16, 17). Indeed, cis-acting elements that potentiate the transcription of Ig and TCR genes also regulate the accessibility of gene segments to recombinase. Deletion of transcriptional enhancers from the TCR, TCR, or the Ig loci significantly reduces both germline transcription and recombination of gene segments in cis- (18, 19, 20, 21). Likewise, deletion of a germline promoter associated with the D1 gene segment severely impairs D1J recombination in thymocytes or in model recombination substrates (22, 23). One apparent exception to enhancer-mediated control of recombinase accessibility was thought to be the IgH locus. Deletion of the only known enhancer near the D_HJ_H region, termed the IgH intronic enhancer (Eµ), only modestly reduced D_HJ_H rearrangements detected in peripheral B cells (24, 25). In contrast, V_HD_HJ_H recombination was severely compromised at the mutant allele.

Two potential explanations have emerged for the minimal effects of Eµ deletions on D_HJ_H recombination. First, prior analyses were performed on splenic B cells that were heterozygous for the mutant IgH allele (Eµ^+/–) (25). These analyses cannot exclude the possibility that Eµ exerts a more profound effect on recombinase accessibility at the D_HJ_H cluster in pro-B cells. Low levels of D_HJ_H recombination at the mutant allele would continue throughout differentiation to the mature B cell stage following productive IgH assembly at the wild-type (WT) allele. Allelic exclusion would effectively block V_HD_HJ_H recombination at the mutant allele in all stages subsequent to pro-B cells. These accumulated D_HJ_H rearrangements in mature B cells would, therefore, mask the true effects of the Eµ knockout on D_HJ_H recombination. Alternatively, a partially redundant element may exist in the D_HJ_H cluster, which could synergize with Eµ to regulate recombinase accessibility. An attractive candidate for this additional element lies adjacent to the most proximal D_H gene segment, termed D_Q52 (see Fig. 1A). This element, termed PD_Q52, exhibits promoter and enhancer activity in pre-B cell models (26). Moreover, PD_Q52 and Eµ colocalize with the only two DNase I hypersensitive sites detected in the D_HJ_H cluster of precursor B cells (27). In prior studies, deletion of the D_Q52 gene segment and a small portion of PD_Q52 had no effect on overall levels of D_HJ_H recombination. Instead, this deletion reduced levels of rearrangements that specifically involved the J_H3 and J_H4 segments (28). Given the architecture of the knockout, however, these studies could not directly address the role of PD_Q52 in directing efficient IgH recombination.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Targeted mutation of the PDQ52 and Eµ elements. A, Schematic depiction of WT and mutant IgH loci generated by homologous recombination (not drawn to scale). Diagrams illustrate the relative positions of the PDQ52 promoter region, the DQ52 and JH gene segments, the intronic enhancer (Eµ), the switch µ region (Iµ-Sµ), and the IgM constant region exons (Cµ). Also indicated are BamHI (B) restriction sites, the relative location of probes A and B used in Southern blotting analysis (B), and the PGK-Neo cassette flanked by loxP sites (triangles). Predicted sizes for BamHI restriction fragments relevant to Southern blotting analysis (B) are shown are thick bars below each schematic. B, Genotypic analysis of mice harboring germline IgH mutations. Southern blotting was performed on BamHI digested tail DNA from mice corresponding to the indicated genotypes (+, WT; N, Neo-containing allele; , Neo-deleted allele). The genomic Southern blots were hybridized to either probe A (left panel) or probe B (right panel). Sizes of the restriction fragments are indicated at the left of each panel.

	Materials and Methods

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Knockout mice

Two targeting vectors were prepared to delete either PD_Q52 alone (P^–E⁺) or PD_Q52 in combination with the 700-bp EcoRI/Xba fragment spanning Eµ and its 5' matrix attachment region (MAR) (P^–E^–; see Fig. 1A). Both targeting vectors were cloned into pBluescript (Stratagene) containing a thymidine kinase gene driven by the PGK promoter. The vectors were linearized with NotI in the polylinker region flanking the 5' homology sequences. The promoter deletion in the P^–E^– construct was generated using PCR by replacing 300-bp upstream of the 5' D_Q52-RSS with a NotI site. A PGK-Neo cassette flanked by two loxP sites was cloned into the NotI site. The 5' homology arm of each vector was derived from C57BL/6 genomic sequences and spanned 6.3 kb upstream of the PD_Q52 deletion. The 3' homology arm contained D_Q52, the J_H cluster, and an additional 1.8 kb of sequences 3' to the Eµ deletion. The P^–E⁺ construct lacked an additional 200 bp of 5' D_Q52 sequences spanning a cluster of XbaI sites that were positioned 600 bp upstream of the PD_Q52 deletion.

Embryonic stem (ES) cells were grown on mouse embryonic fibroblasts and transfected with linearized targeting vectors as described previously (29). Potential recombinants were screened by PCR assays specific for the mutant IgH alleles. All PCR-positive clones were analyzed by Southern blotting to confirm homologous integration of the vectors. Chimeric mice generated from targeted ES cells were mated with C57BL/6 females and germline transmission of the mutated allele was verified by Southern blotting analysis of tail DNA from F₁ offspring (see Fig. 1). The floxed Neo cassette was deleted from targeted alleles by crossing the mutant animals with EIIa-Cre transgenic mice, which express the Cre recombinase at all stages of development (30).

RT-PCR analyses

Total RNA was isolated using TRIzol (Invitrogen Life Technologies) and treated with RNase-free DNase I (37°C, 30 min, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 2 mM MgCl₂). Treated RNA (3 µg) was reverse transcribed using random hexanucleotides (31). cDNA reactions were performed in the presence or absence of reverse transcriptase. The µ0 (31), V_H (32), and Iµ (31) germline transcripts were assessed by PCR assays as described previously. Amplification products were separated on 2% agarose gels, blotted to ZetaProbe membrane (Amersham), and hybridized to the appropriate radiolabeled probes (see PCR primers and probes). The total amount of input cDNA present in each reaction was standardized using an RT-PCR assay that specifically amplifies 5 cDNA sequences (33).

For the D_SP2/D_FL16 RT-PCR assays, 0.5 µg of total RNA was reverse-transcribed for 2 h using Superscript III (Invitrogen Life Technologies) at 50°C, with 100 ng of random hexamers (Amersham Pharmacia). Following reverse transcription, PCR was performed using the following conditions: initial 95°C for 2 min, then 40 cycles consisting of 30 s at 95°C, 30 s at 58°C, 30 s at 72°C, followed by a single 5-min extension at 72°C (for primers, see PCR primers and probes). Amplification products were separated on 3% agarose gels, cloned, and verified by sequence analysis.

PCR assays for IgH rearrangement

DNA extracts were prepared from primary lymphocytes using a cell lysis buffer described previously (31). DNA lysates were prepared at final concentrations of 1 x 10³ cells/µl. The relative levels of D_HJ_H and V_HD_HJ_H rearrangements (31), as well as total DNA content (C) (22), were measured using published PCR assays. To detect D_Q52J_H3 rearrangements, PCR amplifications were performed for 32 cycles consisting of 1 min at 94°C, 1 min at 56°C, and 1.5 min at 72°C, followed by a single 10-min period at 72°C. For detection of germline, unrearranged IgH loci, a PCR assay was used for 30 cycles consisting of 1 min at 94°C, 1 min at 58°C, and 2 min at 72°C, followed by a single 10-min period at 72°C. Amplification products were separated on 2% agarose gels, blotted to ZetaProbe membrane (Amersham), and hybridized to the appropriate radiolabeled probes (see PCR primers and probes).

PCR primers and probes

Primers and probes used in the PCR assays for D_HJ_H and V_HD_HJ_H rearrangements (31), C, -actin (22), V_H (32), µ0, Iµ (31), and 5 (33) have previously been described. Additional primers and probes are as follows. For the D_Q52J_H assay: 5'D_Q52, 5'-GCGGAGCACCACAGTGCAACTG-3'; 3'J₃, 5'-GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG-3'. For the germline IgH PCR assay: 3'J1/1, 5'-GATTGTCACTGTTCCACAGG-3'; 5'J_H1/SJ, 5'-GGACTTTGGGCTGGGTTTGG-3'; 3'J1/2 (probe), 5'-ATGGGATCCCTTCTGCAGCATGCAGAGTGTGGC-3'. For the D_SP2/D_FL16 RT-PCR assays: DFL16.1 forward, 5'-CATACTGGCCAGGGCTTTT-3'; DFL16.1 reverse, 5'-TGGAGCTCAAGAGTCTGCTG-3'; DSP forward, 5'-ACTTGGCAGGGATTTTTGTC-3'; DSP reverse, 5'-TGAAGAGTCTGCTGGGCATA-3'.

FACS analyses

All Abs for cell surface staining were purchased from BD Biosciences. 7AAD was purchased from Molecular Probes (catalog no. A1310). Splenocyte and bone marrow cells from individual, age-matched (6–7 wk) mice were stained with anti-B220-PE (catalog no. 553090) and anti-IgM-FITC (catalog no. 553408) for analysis of total B cell populations, and anti-B220-PE, anti-CD43-biotin (catalog no. 553269) for analysis of precursor B cells. The biotinylated Ab was visualized with streptavidin conjugated to FITC (catalog no. 554057). Flow cytometry was performed on a FACSCalibur instrument (BD Biosciences), and the data were analyzed using CellQuest software. Abs conjugated to magnetic beads for MACS separation were purchased from Miltenyi Biotec.

Isolation of RAG-deficient pro-B cells

Bone marrow cells from RAG-deficient mice harboring either WT, P^–E⁺, or P^–E^– alleles were incubated with a biotinylated anti-B220 Ab (BD Biosciences, catalog no. 553086) for 15 min at 4°C and followed by streptavidin-conjugated MACS beads (Miltenyi Biotec; catalog no. 130-048-101) for 15 min at 4°C. Purified B220⁺ cells were then isolated using the autoMACS sorter (Miltenyi Biotec) and total cellular RNA was isolated for RT-PCR assays as described above.

Isolation of pro-B and pre-B cell populations from bone marrow

Bone marrow cells were isolated from WT, P^–E⁺, P^–E^–, and P⁺E^– mice. The cells were incubated with IgM-conjugated MACS beads (Miltenyi Biotec; catalog no. 130-047-301) for 15 min at 4°C and separated using an autoMACS sorter (Miltenyi Biotec). The IgM^– fraction was incubated with anti-B220-biotin Ab for 15 min at 4°C and then with streptavidin-conjugated MACS beads for 15 min at 4°C. The B220⁺ cells were isolated using the autoMACS sorter and stained with anti-CD43-PE (catalog no. 553271) and anti-AA4.1-allophycocyanin (eBioscience; catalog no. 17-5892-83). The CD43^highAA4.1^high population of pro-B cells and the CD43^lowAA4.1^high population of pre-B cells were sorted on a FACSCalibur instrument (BD Biosciences). The purity of sorted cell populations was >90% as judged by FACS and assays for VJ recombination (data not shown).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation and general characterization of knockout mice

To assess the independent contributions of PD_Q52 and Eµ in controlling the accessibility of IgH gene segments to recombinase, we generated a vector for homologous recombination that replaces 300 bp upstream of the D_Q52 gene segment with a floxed PGK-Neo cassette (Fig. 1A). The specific deletion in this parental vector originates 15 bp upstream of the 5'D_Q52-RSS and spans sequences critical for reported promoter/enhancer activities of PD_Q52 (26, 34). Two targeting vectors were prepared: the first contains the entire Eµ region (P^–E⁺ vector), and the second lacks 700 bp spanning the core Eµ and its 5' MAR (P^–E^– vector) (25, 35). The two targeting vectors were transfected into ES cells and G418-resistant clones were screened for homologous recombination using PCR assays specific for each of the mutant IgH alleles (data not shown). PCR-positive clones were analyzed by Southern blotting to confirm homologous integration of the vectors (Fig. 1B). Chimeric mice were generated by microinjection of targeted ES cell clones into C57BL/6 blastocysts. The chimeras were subsequently mated with C57BL/6 females to obtain germline transmission of the mutant alleles (Fig. 1B). The floxed PGK-Neo cassette was deleted by crossing these animals with mice harboring an EIIA-Cre transgene, which drives expression of Cre recombinase at the earliest stages of embryonic development (30).

Because the functional assembly of IgH loci is a critical component of B lymphocyte development, we first tested whether the P^–E⁺ or P^–E^– mutations impacted this developmental process. Flow cytometric analysis of homozygous mice harboring the PD_Q52 deletion (P^–E⁺) revealed normal proportions of splenic B cells (B220⁺/IgM⁺) when compared with WT control animals (Fig. 2, top panel). Similarly, B cell populations in the bone marrow of P^–E⁺ mice were relatively unaffected by the deletion of the putative promoter element (Fig. 2, middle panel). In sharp contrast, mice homozygous for the P^–E^– allele exhibited a 10-fold decrease in the proportion of B220⁺IgM⁺ cells, indicating a profound block in B cell development. To further define this developmental defect, we stained bone marrow cells for the CD43 and B220 surface markers. The P^–E^– mice possessed a normal complement of B220⁺CD43⁺ pro-B cells but were severely deficient in B220⁺CD43^– pre-B cells (Fig. 2, bottom panel). Thus, the promoter/enhancer-deficient mice exhibit a B cell developmental phenotype consistent with that predicted from Eµ knockout studies. Specifically, impaired V_HD_HJ_H rearrangement is expected to block the transition of precursors from the pro-B to the pre-B cell stage of development (24, 25, 36).

View larger version (65K): [in this window] [in a new window]   FIGURE 2. Impaired B cell development in mice lacking PDQ52 and Eµ. Spleen and bone marrow (BM) cells from 6- to 7-wk-old WT and homozygous mutant mice were analyzed by flow cytometry. Cells were stained with anti-IgM-FITC and anti-B220-PE (top and middle panels) or with anti-CD43-biotin, streptavidin-FITC, and anti-B220-PE (bottom panel). All data were gated for lymphocyte populations. Representative FACS plots from independent analyses of at least four mice for each genotype are shown. The percentages of relevant B cell populations in each tissue are shown in the upper quadrants. No reproducible differences were observed in the cell numbers for spleen or bone marrow from any of the genotypes. Average proportions of B cell populations in each genotype were as follows: BM, CD43+ (pro-B): WT (11.3%), P–E+ (12.8%), P–E– (22.6%); CD43– (pre-B/B): WT (41.3%), P–E+ (39.6%), P–E– (2.2%); IgM+ (B cells): WT (9.5%), P–E+ (7.3%), P–E– (0.6%); spleen, B220+ (B cells): WT (43.1%), P–E+ (46.6%), P–E– (13.1%).

In general, the transcription of germline gene segments is an important correlate of their recombination potential (13, 14, 15, 17). For the IgH locus, two distinct germline transcripts originate from within the D_Q52J_H region. The µ0 transcript initiates upstream of the D_Q52 gene segment and extends through the J_H cluster and Cµ coding exons. This primary transcript is processed to generate a spliced µ0 transcript containing the D_Q52/J_H1 region and Cµ exons (26, 31, 37). The Iµ transcript initiates from multiple sites near Eµ and is spliced from the Iµ mini exon to the Cµ constant region (17, 31) (Fig. 3A). Thus, µ0 and Iµ transcription can be used as an initial readout for chromatin accessibility within the D_Q52J_H region.

View larger version (56K): [in this window] [in a new window]   FIGURE 3. IgH germline transcription in pro-B cells. A, Diagram of the germline DHJH cluster from the mouse IgH locus. DQ52 is a single gene segment, whereas DFL16 and DSP2 represent families of segments spread over a 60-kb array. Primer pairs for PCR assays used to measure DFL16 (A/B), DSP2 (C/D), µ0 (E/G), and Iµ (F/G) transcripts are shown as arrows below the cartoon. B, PCR analysis of Iµ and µ0 germline transcription. Pro-B cells (B220+) were purified from RAG-deficient mice containing either WT or mutant (P–E+ and P–E–) IgH alleles. Total RNA from the purified cells was subjected to RT-PCR assays specific for the transcripts indicated at the left of each panel. Total cDNA levels derived from pro-B cells were measured in each sample using a PCR assay for 5 transcripts. cDNA samples from the RAG-deficient pro-B cell line 63-12, 3T3 fibroblasts, and a derivative of the mature B cell line M12 (5B3) (48 ), were included as positive and negative controls for germline IgH transcription, respectively. The linearity of each assay was confirmed using serial dilutions of cDNA (final six lanes in each panel) derived from the first WT pro-B cell sample (noted by *). PCR amplifications were also performed on RNA samples that were subjected to cDNA reactions lacking reverse transcriptase as a control for genomic DNA contamination (-RT). C, Pro-B cell samples described in B were subjected to PCR assays for germline transcripts derived from the DFL16 and DSP2 families of IgH gene segments. Assay linearity was confirmed with serial 2-fold dilutions of each sample.

In contrast to the single mutation, dual deletion of PD_Q52 and Eµ led to substantial reductions in the steady-state levels of both Iµ and µ0 germline transcripts (Fig. 3B, top and middle panels). Control experiments verified that purified cells from each IgH genotype expressed similar levels of the pro-B cell-specific 5 gene (Fig. 3B, bottom panel). Similar results were obtained with pro-B cells from mice lacking only the core Eµ element (38), indicating a dominant role for the enhancer in this transcriptional defect at the D_Q52J_H cluster. We conclude that the PD_Q52 deletion in the P^–E^– allele is functionally neutral; the primary phenotypes displayed at this mutant IgH locus are largely attributable to the accompanying enhancer deletion.

To further define the effect of Eµ on chromatin accessibility throughout the entire D_HJ_H cluster, we measured germline transcripts from the D_FL16 and D_SP2 gene families, which lie from 10 to 80 kb upstream of the D_Q52 segment (Fig. 4) (39). Consistent with µ0 and Iµ expression data, D_FL16 and D_SP2 germline transcription was unaffected by the PD_Q52 deletion (Fig. 3C). However, mice homozygous for the P^–E^– mutation exhibited at least a 5-fold reduction in steady-state levels of the D_FL16 and D_SP2 germline transcripts. Together, these results indicate that efficient germline expression of the entire D_HJ_H cluster is regulated by the IgH intronic enhancer and further suggest that Eµ is important for controlling long-range chromatin accessibility of this region within the IgH locus.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Inhibition of DHJH recombination in pro-B cells from Eµ-deficient mice. A, Schematic depiction of PCR assays for DHJH rearrangement (top). Arrows denote the relative position of PCR primers (31 ). Purified pro-B cells from the indicated IgH genotypes were subjected to the DHJH PCR assay, and the results are shown at the bottom. Samples from two experiments using sorted cells from independent mice are shown for each genotype. Amplification products corresponding to germline IgH alleles (GL), as well as DHJH1 (J1), DHJH2 (J2), and DHJH3 (J3) rearrangements are denoted at the left. Control PCR assays for total DNA content (C) are shown in the bottom panel. The linearity of each assay was confirmed using serial dilutions of unsorted WT bone marrow (BM) and WT pro-B cells shown in lane 1 (1/2, 1/5, and 1/10). Genomic DNA from the RAG-deficient pro-B cell line 63-12 was included as a negative control. B, Diagrammatic representation of the PCR assay for measuring retention of germline IgH alleles (top). Pro-B cell DNA from WT, P–E+, P–E–, and P+E– mice were analyzed for levels of germline IgH alleles (GL) and total DNA (C). Generation of P+E– mice was described in Ref.38 . Titrations were performed on DNA from unsorted WT bone marrow (BM) and from the RAG-deficient pro-B cell line 63-12 (final eight lanes).

Prior analyses of IgH gene assembly in Eµ knockout mice were performed on mature B cells that were heterozygous for an enhancer-deficient allele (Eµ^+/–). These studies suggested that deletion of Eµ has only a modest effect on D_HJ_H recombination (2-fold) (24, 25). Because these experiments used endpoint B cells, the possibility remained that Eµ may have a more profound impact on the initiation of D_HJ_H recombination in pro-B cells. Specifically, the D_HJ_H cluster may continue to rearrange in later stages of B cell development that express V(D)J recombinase (i.e., pre-B and emerging B cells). This scenario would also explain the profound block in V_HDJ_H rearrangements observed for Eµ^+/– B cells, because allelic exclusion mechanisms would suppress this process in pre-B and emerging B cells.

To assess whether Eµ or PD_Q52 function to regulate D_HJ_H recombination in pro-B cells, we sorted this precursor population from the bone marrows of WT, P^–E⁺, and P^–E^– mice using flow cytometry (see Materials and Methods). Genomic DNA from the purified cells was subjected to PCR assays using a degenerate set of oligonucleotide primers that recognize sequences directly 5' of 12 D_H gene segments (Fig. 4A, primer A) (31), a primer that recognizes the 5' D_Q52-RSS (primer B), and a downstream primer specific for a sequence 3' to the J_H3 gene segment (primer C). Using this assay, impaired D_HJ_H recombination would be manifest as both a reduction in the levels of detectable rearrangement products and as an enhanced retention of the germline IgH allele. As shown in Fig. 4A, deletion of PD_Q52 had no detectable effect on the loss of germline IgH alleles and exhibited normal levels of D_HJ_H rearrangement when compared with WT pro-B cells. Thus, similar to germline transcription, PD_Q52 is dispensable for normal D_HJ_H rearrangement. In contrast, dual deletion of PD_Q52 and Eµ resulted in a dramatic retention of germline IgH alleles, indicating a profound block in D_HJ_H rearrangement. Similar data were obtained using PCR assays that specifically detect rearrangements involving only the D_Q52 gene segment or that monitor recombination of all four J_H segments (data not shown).

To more accurately measure the effects of these cis-acting elements on the efficiency of D_HJ_H recombination, we performed a second PCR assay, which detects only the retention of germline IgH loci in DNA from purified pro-B cells. Data from these analyses were normalized for total DNA content using a separate PCR assay specific for germline sequences in the Ig constant region exon (C) (22). As shown in Fig. 4B, pro-B cells from WT and P^–E⁺ mice exhibited a comparable loss of germline IgH alleles, indicating that IgH rearrangements were unaffected by the PD_Q52 deletion. In contrast, we observed a profound increase in the retention of germline IgH alleles in pro-B cells harboring the P^–E^– mutation. When normalized for DNA content (C), semiquantitative analysis of these PCR data indicated an increase in the retention of germline IgH loci in P^–E^– cells (IgH:C ratio 1.7) that ranged from 5- to 10-fold higher when compared with normalized signals in WT cells from C57BL/6 littermates (IgH:C ratio 0.25). We also examined pro-B cell samples from mice that harbored a deletion of only the Eµ element (P⁺E^–) (38) and its corresponding WT littermates (129 strain). Unlike the P^–E^– mutation, which lacked Eµ and its 5' MARs, only the core Eµ element was deleted in the P⁺E^– mutation. Pro-B cells from the WT 129 strain retained higher levels of germline IgH alleles compared with their C57BL/6 counterparts (IgH:C ratios 1.1 and 0.25, respectively). However, the single Eµ knockout displayed an 5-fold increase in retention of their germline IgH alleles relative to the 129 pro-B cells (IgH:C ratio 4.8). Together, these data indicate that Eµ plays a more significant role in the regulation of D_HJ_H recombination than was previously appreciated.

Pro-B cells purified from adult bone marrow are a mixed population of cells with regards to the genetic configuration of the IgH locus. The cells used in our analyses undergo both D_HJ_H and V_HDJ_H recombination. One complicating factor we considered is that rearrangements involving V_H segments will eliminate annealing sites for primers used in D_HJ_H rearrangement assays. In contrast to pro-B cells, a significant portion of thymocytes contain D_HJ_H rearrangements but are blocked from completing V_HDJ_H recombination (40). Thus, thymocytes offer an independent cell system to directly test the effects of PD_Q52 and Eµ on D_HJ_H rearrangement efficiencies without the complications associated with V_HDJ_H recombination. As shown in Fig. 5, D_HJ_H rearrangement was unaffected in thymocytes from P^–E⁺ mice, which lack only the PD_Q52 region. Dual deletion of PD_Q52 and Eµ or a single deletion of only the core enhancer both led to a dramatic reduction in the levels of D_HJ_H rearrangement detected in thymocytes (>20-fold decrease). These findings further demonstrate that, although PD_Q52 is dispensable, Eµ plays an important role in controlling both germline transcription and recombination of the D_HJ_H cluster.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. Eµ deletions completely inhibit DHJH rearrangement in thymocytes. Whole thymocyte DNAs from the indicated IgH genotypes were subjected to PCR assays for either DQ52JH (upper panel) or overall DHJH recombination (lower panel). The relative positions of amplification products corresponding to germline IgH alleles (GL), or alleles containing DHJH1 (J1), DHJH2 (J2) or DHJH3 (J3) rearrangements are shown at the left. Each set shows a serial dilution of one sample (1/2, 1/5, and 1/10). Identical results were obtained using thymocyte DNA from multiple mice of each genotype (data not shown). Refer to Fig. 4 legend for additional details.

Prior studies using mature B cells from Eµ^+/– animals indicated that deletion of the intronic enhancer severely impairs V_HD_HJ_H rearrangement (24, 25), suggesting that V_H gene segments are either directly or indirectly regulated by Eµ. However, our new data derived from pro-B cells suggest that the block in V_HD_HJ_H rearrangement may be a secondary effect resulting from reduced D_HJ_H recombination. To address the possibility that the enhancer is directly regulating V_H accessibility, we studied the effects of P_DQ52 and Eµ deletions on V_H rearrangement and germline transcription. We first used a published PCR assay to examine V_HD_HJ_H rearrangements in sorted pro-B cells (31) (Fig. 6A). Using this assay, we found that PD_Q52 is dispensable for V_HD_HJ_H rearrangement. In keeping with prior Eµ knockout studies, dual deletion of PD_Q52 and Eµ resulted in a marked reduction of V_HD_HJ_H rearrangement. Similar results were obtained from PCR assays using primers specific for the rearrangement of three major V_H families (V_HJ558, V_HQ52, and V_H7183), which are spread throughout the entire V_H cluster (data not shown). Thus, inhibition of V_HD_HJ_H recombination in these animals is not due to a regional loss of recombinase accessibility at only a subset of V_H gene segments.

View larger version (42K): [in this window] [in a new window]   FIGURE 6. Deletion of the intronic enhancer affects VHDHJH recombination independent of germline VH transcription. A, Schematic depiction of PCR assays for VHDHJH rearrangement and VH germline transcription (top). For VHDHJH recombination, a mixture of degenerate primers that recognize the three major VH families (VH558, VHQ52, and VH7183) were used in conjunction with a primer specific for the JH3 gene segment (31 ). The relative position of amplification products corresponding to VHDHJH1 (J1), VHDHJH2 (J2), and VHDHJH3 (J3) rearrangements are indicated at the left. Controls and sample titrations of the first WT sample are described in the legend for Fig. 4. B, Germline VH transcription. Total cellular RNA was isolated from B220+ bone marrow cells (pro-B) from RAG-deficient mice that were homozygous for the indicated IgH alleles. The RNA was subjected to RT-PCR assays for VH germline transcription using degenerate primers for three VH gene families (32 ). PCR products were blotted and hybridized with radiolabeled oligonucleotide probes that recognize specific sequences in each of these VH families. Total cDNA levels were controlled in each sample using a PCR assay for the pro-B cell-specific 5 transcript. Refer to Fig. 3 legend for a description of additional controls. The linearity of each assay was confirmed using serial dilutions of cDNA (final five lanes in each panel) derived from the first sample denoted by *.

	Discussion

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The targeted deletion of transcriptional promoters and enhancers at most Ag receptor loci represses chromatin accessibility at linked gene segments and blocks their assembly by V(D)J recombinase (18, 19, 20, 21). In contrast, deletion of the IgH intronic enhancer (Eµ) had a minimal impact on primary D_HJ_H rearrangements, but significantly reduced levels of V_HD_HJ_H recombination when assessed in peripheral B cells (24, 25). One plausible explanation for this phenotype is the existence of an unidentified control element near the D_HJ_H cluster that can compensate for the loss of Eµ. A prime candidate for the compensatory element is PD_Q52, which has promoter/enhancer activity in reporter assays and, together with Eµ, is the only DNase hypersensitive region within the D_HJ_H cluster (26, 41). An alternative explanation is that Eµ may have a more substantial role in the regulation of D_HJ_H recombination but continued rearrangement of the mutant allele during B cell development masks its full impact when assessed in peripheral B cells (25).

To test both of these possibilities, we generated mice with targeted deletions of PD_Q52 and Eµ. Our experimental results demonstrate that: 1) the PD_Q52 element defined by reporter assays has minimal impact on IgH germline transcription and rearrangement; 2) when analyzed in pro-B cells or thymocytes, deletion of Eµ significantly impairs D_HJ_H recombination; 3) Eµ plays an important role in the regulation of germline transcripts throughout the D_HJ_H cluster; and 4) Eµ is dispensable for transcription of germline V_H gene segments. Together, these findings indicate that Eµ is an important component of the mechanisms controlling recombinase accessibility at the D_HJ_H cluster in pro-B cells. The defect in V_HD_HJ_H recombination is likely a downstream consequence of impaired D_HJ_H recombination at Eµ-deficient alleles (see below).

A surprising result from our knockout studies is that deletion of the previously defined PD_Q52 element has no effect on germline transcription of the D_HJ_H cluster (Fig. 3). In this regard, an independent deletion of the D_Q52 gene segment and a smaller portion of its 5' region also retained germline D_HJ_H transcription (28). These findings indicate that additional promoter and/or enhancer activity must reside upstream from "PD_Q52" (26). Additional studies are required to identify these novel elements and to determine their role in the regulation of chromatin accessibility at the D_HJ_H cluster. We conclude that defects in germline transcription and D_HJ_H recombination arising from the dual P^–E^– mutation are largely or solely due to the absence of Eµ rather than PD_Q52.

To directly assess the effects of Eµ deletions on primary D_HJ_H recombination, we analyzed purified pro-B cells, the developmental stage that initiates IgH gene assembly. Pro-B cells from enhancer-deficient mice exhibit at least a 5- to 10-fold increase in their retention of germline IgH alleles when compared with WT counterparts (Fig. 4B). These data are fully consistent with a contemporaneous study showing that B cell hybridomas from mice heterozygous for a deletion of the core Eµ element have an elevated retention of the germline IgH configuration only at mutant alleles (38). When normalized for DNA content and compared with its corresponding WT strain, pro-B cells from these P⁺E^– mice had slightly lower levels of germline IgH alleles than pro-B cells harboring the P^–E^– mutation (Fig. 4B and data not shown). Thus, PD_Q52 may possess a modest compensatory function for regulation of the D_HJ_H cluster in the absence of Eµ. However, this interpretation is difficult to resolve for two reasons: 1) the P^–E^– allele harbors a more extensive deletion of the intronic enhancer, which includes the 5' MARs; and 2) the P^–E⁺ and P⁺E^– alleles were bred into distinct genetic backgrounds (C57BL/6 and 129, respectively).

Notwithstanding these uncertainties, a dominant role for Eµ in the regulation of accessibility at the D_HJ_H cluster is supported by additional evidence emerging from our studies. Deletion of the enhancer together with PD_Q52 affects germline transcription of D_H gene segments throughout the entire cluster, which spans nearly 80 kb (Fig. 3). Moreover, D_HJ_H rearrangement is completely blocked in thymocytes from Eµ-deficient mice (Fig. 5). Collectively, these results argue that Eµ serves as a long-range control element to exert a positive influence on efficient recombination throughout the entire D_HJ_H cluster. This influence is critical to facilitate efficient D_HJ_H recombination and to guide subsequent stages of B cell development.

Consistent with prior studies of mice containing B cells that were heterozygous for Eµ-deleted alleles, we observe a profound block in V_HD_HJ_H recombination in pro-B cells from the P^–E^– mice. A possible interpretation of these results is that Eµ directly regulates accessibility of V_H gene segments, which are located at least 100 kb upstream from this enhancer. However, we find that germline transcription of the V_H cluster is unaffected by the enhancer deletion (Fig. 6). Collectively, our studies support an alternative interpretation–the block in V_HDJ_H rearrangement is largely a secondary defect stemming from a substantial reduction in the formation of substrates containing a requisite D_HJ_H junction. This interpretation is fully consistent with data from enhancer deletions at the TCR locus, which inhibit VDJ rearrangement as a consequence of a loss in chromatin accessibility at the DJ but not at the V clusters (42).

Although the primary defect in Eµ-deficient pro-B cells is inhibition of D_HJ_H recombination, the reductions in subsequent V_HDJ_H rearrangement are even more pronounced. We hypothesize that the further reduction in V_HD_HJ_H junctions may be a consequence of the "stalling-out" that must occur in B cell development as a result of Eµ deletions. Precursor cells stuck at the pro-B stage may continue to accumulate D_HJ_H joins at a lower rate, but given the limited lifespan of these cells, the remaining window of time for V_HD_HJ_H recombination may be extremely narrow. Consistent with this possibility, we analyzed IgH rearrangements in precursor B cells from fetal liver, which undergo a synchronous wave of either D_HJ_H (day 14) or V_HD_HJ_H recombination (day 16). In these cells, we find that D_HJ_H and V_HD_HJ_H recombination are compromised to an equal extent when Eµ is deleted (data not shown). Despite these findings, we cannot rule out an auxiliary role for Eµ in the complex mechanisms that regulate efficient V_HD_HJ_H recombination, including contraction of the IgH locus or changes in chromatin modifications at V_H gene segments (41, 43, 44).

Although deletion of Eµ clearly affects chromatin accessibility throughout the D_HJ_H cluster, these gene segments continue to rearrange with a modest efficiency. The residual recombination at Eµ-deficient alleles indicates the existence of additional cis-elements involved in the control of D_HJ_H accessibility. Our findings strongly suggest that the function of this unidentified element must be restricted to pro-B cells because D_HJ_H recombination is completely abolished in Eµ-deficient thymocytes (Fig. 5). Based on prior studies, several candidates can be envisioned for the additional regulatory element(s): 1) The region upstream of PD_Q52. The retention of germline D_HJ_H transcripts in P^–E⁺ mice strongly suggests additional promoter activity within this region. Analogous to the TCR locus (45), deletion of the complete germline promoter may abrogate D_HJ_H rearrangement. 2) The 3' C region contains four enhancers that can regulate promoters over extremely large distances (>200 kb) (46). This global effect may partially compensate for the loss of Eµ function, leading to a partial activation of germline transcription and D_HJ_H recombination. 3) A defined region between the C and C3 coding exons (E-3) in the human IgH locus has been shown to potentiate transcription from transgenic reporter genes in mouse precursor B cells. This putative enhancer, which may have a murine homolog, also functions cooperatively with Eµ in reporter assays (47). The mice reported in the present study, which harbor deletions in the two known D_HJ_H regulatory elements, will be important reagents for deciphering the complex interplay of promoters and enhancers that activate IgH gene assembly and trigger the B cell developmental program.

	Acknowledgments

 
We thank C. Edson and O. Osipovich for their contributions to the initial stages of this project. We are also grateful to F. Alt and colleagues for providing core Eµ knockout animals and sharing unpublished data.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the National Institutes of Health (P01 HL68744 and CA100905 to E.M.O.; T32 CA09385 to R.A.) and a Cancer Center Support Grant (P30 CA68485, Vanderbilt-Ingram Cancer Center).

² Address correspondence and reprint requests to Dr. Eugene M. Oltz, Department of Microbiology/Immunology, Vanderbilt University Medical School, Nashville, TN 37232. E-mail address: eugene.oltz{at}vanderbilt.edu

³ Abbreviations used in this paper: RSS, recombination signal sequence; MAR, matrix attachment region; ES, embryonic stem; WT, wild type.

Received for publication September 2, 2005. Accepted for publication December 1, 2005.

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