Enhancers Located in Heavy Chain Regulatory Region (hs3a, hs1,2, hs3b, and hs4) Are Dispensable for Diversity of VDJ Recombination*

Background: We examined the effect of deletion of the heavy chain regulatory region (RR) on VDJ recombination in B-cells. Results: V, D, and J usage is unaffected by the absence of the IgH RR. Conclusion: The IgH RR is dispensable for V(D)J diversity. Significance: This region only orchestrates IgH locus activity during late stages of B-cell differentiation. V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple IgH cis-regulatory elements control B-cell ontogeny. IGCR1 (intergenic control region 1), the DQ52 promoter/enhancer, and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination, whereas there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here, we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B-cell differentiation. In contrast, the earliest antigen-independent steps of B-cell ontogeny would be under the control of only the upstream Cmu elements of the locus.

Lymphopoiesis is coupled with programmed accessibility of Ig genes to transcription and to several major transcription-dependent DNA remodeling events (1,2). During the antigenindependent step of B-cell ontogeny, the V(D)J recombination process within the IgH locus allows the assembly and expression of the functional heavy chain gene (3). Multiple cis-regulatory elements located 5Ј and 3Ј of constant genes control B-cell ontogeny. The intronic 5Ј E element is reported as a master control element of V(D)J recombination (4). Deletion of the 5Ј DQ52 element leads to a minor effect on V(D)J recombination and altered D gene usage (5,6). Recently, IGCR1 (intergenic control region 1), which lies between the V H and D clusters, was reported to promote rearrangement of distal rather than D H -proximal V H segments (7,8). The IgH 3Ј regulatory region (3ЈRR) 4 that encompasses the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 has been reported as the master element controlling class switch recombination (2,9) and is also important for oncogene deregulation in B-cell lymphomagenesis (10,11). Whereas physical chromosomal interactions have been reported in pro-B-cells between the 5Ј E enhancer and the 3ЈRR (12,13) and whereas GFP transgenic mice highlighted that combination of 5Ј E and the 3ЈRR mimics the B-specific endogenous expression pattern of IgH genes from pro-B-cells to mature B-cells (14,15), the potential contribution of the 3ЈRR to V(D)J recombination control has never been studied. Although bacterial artificial chromosome transgenes have been used to explore the role of the 3ЈRR in class switch recombination and somatic hypermutation (16,17), they are inappropriate to test its role in V(D)J recombination. Because antigen receptor gene accessibility studies need the context of endogenous loci, only animals with genomic deletions are suitable for such investigations. To clarify the role of the 3ЈRR in V(D)J recombination, we analyzed 3ЈRR-deficient mice lacking the whole 30-kb extent (from hs3a to hs4) of the 3ЈRR; we recently reported a severe class switch recombination defect and an associated IgH transcription defect at the plasma cell stage in these mice (9).

MATERIALS AND METHODS
Mice-Generation of 3ЈRR-deficient mice has been described previously (7).
Amplification Procedures-Genomic DNA was extracted from the bone marrow CD25 ϩ population (including precursor B-cell fractions CЈ and D) (18) from 3ЈRR-deficient and 129 WT mice (three groups of five mice). Repertoire diversity was measured using the ImmunЈIg test (ImmunID Technologies, Grenoble, France). Multiplex PCR was performed as described (19) using an upstream primer specific to all functional members of a given V family and a downstream primer specific to a * This work was supported in part by grants from the Ligue contre le Cancer (Comité Départemental de la Haute-Vienne, de la Creuse, et de la Corrèze), the Conseil Régional du Limousin, and Agence Nationale de Recherche (Projets Blanc 2011). 1  given J segment. This assay allows the simultaneous detection and resolution of 92 V-J rearrangements. For semiquantitative analysis, PCRs were stopped at their exponential step.
Cloning and Sequencing-Genomic DNA from B-splenocytes was amplified by PCR using the following primers: forward, 5Ј-GCGAAGCTTARGCCTGGGRCTTCAGTG-AAG-3Ј (complementary to the V H J558 segment); and backward, 5-AGGCTCTGAGATCCCTAGACAG-3Ј (found in the J H4 segment). PCR products were cloned into the pGEM-T Easy vector system (Promega) and sequenced using an ABI PRISM automated laser fluorescent sequencer (PerkinElmer Life Sciences).

3ЈRR-deficient IgH Allele Is Normally Used in Heterozygous
IgH a ⌬3ЈRR /b WT Mice-3ЈRR-deficient mice have been described previously (9). Because the disruption was carried out in a 129 embryonic stem cell line, it resulted in a mutated a allotype IgH locus. Mice were thus bred with C57BL/6 animals to derive heterozygous IgH a ⌬3ЈRR /b WT mice. Cell cytometry analysis of bone marrow cells from a ⌬3ЈRR /b WT mice with anti-IgM allotype-specific antibodies indicated that similar amounts of B-cells expressed either the a or b allotype (Fig. 1). This can be taken as an indication that the 3ЈRR-deficient allele underwent V(D)J recombination at a rate similar to the WT allele.

V(D)J Usage Is Not Affected in 3ЈRR-deficient Mouse
Pre-B-cells-We investigated V(D)J usage in 3ЈRR-deficient mice compared with 129 WT mice. Pre-B-cells express a broad random repertoire of V H genes, whereas mature B-cells dominantly express only some sets of V H genes due to ligand selection (21). To bypass a bias generated by antigen selection, V(D)J usage was investigated in the bone marrow CD25 ϩ population (including mostly fraction CЈ and D pre-B-cell precursors) (18). Ninety-two PCR experiments were done, exploring a wide diversity of V H segments (from the most upstream to the most downstream) and the four J H segments. The diversity of rearrangement (ratio of observed rearrangement to theoretical rearrangement) was similar (p ϭ 0.8, Mann-Whitney U test) between 3ЈRR-deficient mice (83.3 Ϯ 3.8%, mean Ϯ S.E.) and 129 WT mice (85.9 Ϯ 2.7%) (Fig. 2A). The J 1-4 usage was not affected by the 3ЈRR deletion (Fig. 2B). Finally, the vast majority (21/23) of V segments were used in a similar manner between 3ЈRR-deficient and WT mice (Fig. 2C). Taken together, these results suggest that V(D)J rearrangements normally occurred in bone marrow CD25 ϩ B-cells of 3ЈRR-deficient mice, and there was no indication of any gross alteration in the V(D)J recombination process.

V(D)J Junctions Have Normal Structures in 3ЈRR-deficient
Mice-To appreciate the potential effect of 3ЈRR deletion on D usage, V(D)J junction sequences from CD25 ϩ pre-B-cells from 3ЈRR-deficient and 129 WT mice were amplified, cloned, and sequenced. Of 95 sequences from WT mice and 157 sequences from 3ЈRR-deficient mice, we found that there was no significant difference with regard to the percentage of functional sequences, CDR3 (complementarity-determining region 3) length, and number of N nucleotides inserted at the V-D-J junc-  MARCH 9, 2012 • VOLUME 287 • NUMBER 11 tions (Table 1). D usage in a 3ЈRR-deficient mouse appeared similar compared with a WT mouse. The D segments were used with frequencies that did not differ from WT mice, including the most upstream (DFL16,3) and the most downstream (DQ52) segments. There was thus no indication of any gross alteration in the DJ recombination process. The extent of N insertions was normal in 3ЈRR-deficient animals, suggesting that these rearrangements normally occurred at a stage where pro-B-cells express terminal deoxynucleotidyltransferase activity. Similar results were found after investigation of V(D)J junction sequences from B-splenocytes (i.e. after interaction with cognate antigens) from homozygous 3ЈRR-deficient mice (89 sequences) and WT mice (100 sequences) ( Table 2).

V(D)J Diversity in IgH 3RR-deficient Mice
Conclusion-Despite its physical interaction with E at early stages of B-cell development and, de facto, its eventual link to the proximity of rearranging V, D, and J segments (14,15), it appears that the 3ЈRR is dispensable for the process of V(D)J recombination in B-cell progenitors. Although the 3ЈRR con-trols class switch recombination in differentiated B-cells (9), it appears to be dispensable for the process of V(D)J recombination in B-cell progenitors. These results extend those obtained with hs3b/hs4-deficient mice (22). They clearly contrast with those obtained with E-, DQ52-, and IGCR1-deficient animals (4,8) and suggest that, among cis-activating elements of the IgH locus, those located in the 5Ј part of the locus are fully sufficient for the optimal completion of V(D)J recombination.