Complete IgHC gene rearrangement occurs only in B cells in a stage-specific and ordered manner. We used gene targeting to reposition a distal VH gene segment to a region just 5′ of the DH gene cluster and found its activation to be highly dependent on the chromosomal domain within which it resides. The targeted VH gene segment rearranged at a higher frequency than its endogenous counterpart, its rearrangement was no longer ordered, and its ability to be silenced by allelic exclusion was lost. Additionally, the targeted VH gene segment lost lineage specificity, as VDJH rearrangement was observed in thymocytes. These data suggest that locus contraction, mimicked by proximal targeting, can override any regulation imposed by DNA sequences immediately surrounding VH gene segments.
V(D)J recombination underlies the remarkable diversity of antigen receptors in the immune system (for review see reference 1). A common recombinase dependent on the lymphocyte-specific gene products RAG1 and RAG2 recognizes and cleaves pairs of conserved recombination signal sequences (RSSs), which flank all Ig and TCR V, D, and J gene segments. Components of the nonhomologous end-joining double-stranded DNA (dsDNA) break repair system then catalyze the formation of coding exons by ligating the pair of broken coding ends to one another. Because RSSs at each of the seven rearranging loci (IgHC; κ- and λLC; and TCRα, -β, -γ, and -δ chains) are recognized by the same recombinase machinery, the cell-type and stage-specific regulation of rearrangement is thought to rely on the accessibility of the recombinase to specific rearranging loci within chromatin structure (2, 3).
V(D)J recombination is regulated in three types of ways. First, rearrangement occurs in a lineage-specific manner. Ig and TCR genes rearrange completely only in developing B and T cells, respectively. Second, rearrangement is ordered within a lineage with IgHC or TCRβ locus rearrangement preceding IgLC or TCRα locus rearrangement in B and T cells, respectively. In addition, D-to-J rearrangement precedes V-to-DJ rearrangement in both IgHC and TCRβ loci. Finally, recombination in the IgHC and TCRβ loci are subject to allelic exclusion: the observation that each developing lymphocyte assembles only one functional IgHC or TCRβ chain gene, contributing to the clonotypic specificity of antigen recognition.
The IgHC locus has been extensively studied in an attempt to decipher the molecular basis of regulated V(D)J recombination. As noted in the previous paragraph, VH-to-DJH rearrangement invariably follows DH-to-JH rearrangement (4). Direct VH-to-DH rearrangement is not observed, even on alleles with a targeted deletion of the JH cluster of gene segments (5). Perhaps more remarkably, a VH gene segment will bypass intervening germline DH segments to join to a partially rearranged DJH segment. The DH-to-JH step in IgHC gene assembly is not lineage specific. DJH alleles are found in up to 40% of T cells, but complete VDJH alleles are not seen in these cells (6). In addition, unlike VH-to-DJH rearrangement, DH-to-JH rearrangement is not subject to allelic exclusion. IgHC transgenic mice contain endogenous DJH- but minimal VDJH-rearranged alleles (7, 8).
Several trans-acting factors and signaling pathways have been implicated in the regulation of VH-to-DJH rearrangement. Mice deficient in the transcription factor Pax5, the histone methyltransferase Ezh2, or the IL-7Rα chain show defects at this step of IgHC gene assembly (9–13). In each of these instances, the defect is greater for the more distal VH gene segments, suggesting that long-range chromosomal interactions may play an important role in this regulation. This idea is consistent with the results of fluorescent in situ hybridization experiments showing developmentally regulated chromosomal compaction or looping of distal VH genes into juxtaposition with the DH-JH end of the IgHC locus (14–17). STAT5 (activated by IL-7Rα signaling), Pax5, and Ezh2 have each been shown to localize to VH region sequences in vivo (11, 18, 19). Remarkably, forced expression of Pax5 in developing T cells results in the activation of VH-to-DJH rearrangement and partial locus compaction in the “wrong” lineage (14, 20).
Rearrangement within the IgHC locus is influenced in cis by the intronic heavy-chain enhancer. Targeted deletion of this element results in a moderate decrease in DH-to-JH rearrangement but a near-complete absence of VH-to-DJH rearrangement (21–23). Perhaps surprisingly, deletion of the most JH-proximal DH gene, DQ52, along with a promoter 5′ of this gene segment that is responsible for germline transcription of the JH cluster of gene segments, has little effect on IgHC rearrangement (23, 24).
In the present paper, we describe experiments aimed at distinguishing whether DNA sequences immediately surrounding VH gene segments are sufficient for the proper regulation of VH-to-DJH rearrangement or whether the regulation of rearrangement depends on the chromosomal position and the context of a VH gene segment. We found that targeting a distal VH gene segment to a region ∼1 kb 5′ of DFL16.1 caused it to recruit activating chromatin modifications, to rearrange more frequently than its endogenous counterpart, to rearrange directly to unrearranged DH gene segments, to violate allelic exclusion, and to lose lineage specificity. We conclude that chromosomal position profoundly affects the regulation of VH gene segment rearrangement.
Targeted insertion of a distal VH gene segment into the 5′ of D region of the IgHC locus
To test to what extent chromosomal proximity contributes to the regulation of VH-to-DH rearrangement, we used homologous recombination in embryonic stem cells to target a distal VH558 family gene segment along with ∼1.3 kb of upstream promoter sequence and ∼500 bp of downstream sequence to a region ∼500 bp 5′ of DFL16.1 (Fig. 1). The distance from DFL16.1 to the RSS of the targeted VH gene (termed VH-KI) is ∼1 kb. Both the conceptual translation of this VH gene segment and its RSS closely match the consensus for the VH558 gene family (unpublished data). In addition, its promoter sequence contains the canonical octamer binding site. Presumably, an identical copy of this VH gene, referred to as its endogenous counterpart, lies in the distal region of the IgHC locus, although the existence of a VH gene with this exact sequence was not demonstrated in a paper on the sequence of the IgHC locus from another mouse strain (25).
VH-KI is frequently rearranged and expressed in knock-in mice
To measure the frequency of VH-KI rearrangement, we took advantage of the fact that the targeted VH gene possesses an SspI restriction endonuclease site that only one other functional VH gene is predicted to have (Fig. 2 A). We amplified complementary DNA (cDNA) synthesized from bone marrow, spleen, CD4+ CD8+ (double-positive [DP]) thymocyte, and IL-7–dependent pro–B cell culture RNA from wild-type and homozygous VH-KI mice with a degenerate VH gene primer that is complementary to the leader sequence of most VH558 family genes paired with a primer complementary to an IgHC constant region exon. RT-PCR products were subjected to digestion with SspI to assess what fraction of the products represent transcription of a rearranged VH-KI gene segment. The wild-type samples had minimal cleavage from the contribution of endogenous VH genes, whereas the fraction of cleavable product in the targeted animals was significant (Fig. 2 B), implying that the targeted VH gene segment rearranges very frequently. In IL-7–dependent pro–B cell cultures, cells are not subject to selective pressure for pre-BCR assembly. In this setting, about half of the total VH558 family rearrangements involve the targeted VH gene segment, and this does not appear to differ in primary cells from bone marrow and spleen of these animals. Thus, IgHC rearrangements involving the VH-KI gene segment can apparently undergo positive selection during B cell development and contribute to the B cell repertoire in knock-in mice. As expected, the DP thymocytes do not produce spliced rearranged transcripts.
The targeted VH gene segment recruits activating chromatin modifications in thymocytes
Because the targeted VH-KI gene segment was inserted within a region containing developmentally regulated histone modifications in cell lines (Fig. 3 A) (26), we proceeded to compare the histone modifications surrounding the targeted VH gene with those in the region 5′ of DFL16.1 on the unaltered allele in heterozygous VH-KI animals. We performed chromatin immunoprecipitations (ChIPs) on VH-KI heterozygous RAGnull bone marrow cultured in IL-7 and on VH-KI heterozygous RAGwt thymocytes using antibodies that recognize H3 acetylation and H3K4 dimethylation. We used RAGnull bone marrow to ensure that sufficient germline sequence would be available for the PCR reaction. The primer set called 5′V amplifies a region ∼2 kb upstream of the VH gene segment sequence, and 3′V amplifies a region ∼400 bp downstream of the targeted VH gene segment RSS (Fig. 3 B). Predictably, we found that the targeted VH gene segment is modestly enriched for H3 acetylation and significantly enriched for H3K4 dimethylation in pro–B cells. To our surprise, we found that the 3′ end of the VH-KI insertion, but not the unperturbed allelic region 5′ of DFL16.1, was enriched for H3K4 dimethylation in thymocytes (Fig. 3 C). Thus, the VH gene segment is capable of recruiting histone modifications not otherwise found 5′ of DFL16.1 in unperturbed thymocytes.
VH-KI undergoes rearrangement in DP thymocytes
Given our observation that the targeted VH gene segment can recruit H3K4 methylation in T cells, we went on to ask whether the lineage specificity of VH-to-DJH rearrangement was likewise perturbed by repositioning this VH gene segment. We purified wild-type, heterozygous, and homozygous VH-KI genomic DNA from bone marrow, spleen, and sorted (>99% pure on reanalysis; unpublished data) DP thymocytes and used PCR to detect VH-to-DJH and DH-to-JH rearrangements. As expected, we detected rearrangement of DFL16.1-to-JH segments in both wild-type and mutant samples. In wild-type samples, VH558-, VH7183-, and VHQ52-to-DJH rearrangement was limited to the bone marrow and spleen (Fig. 4). In contrast, abundant VH-KI–to–DJH rearrangements were observed in all three tissues, including DP T cells from both VH-KI heterozygous and homozygous animals. This demonstrates that T cells are capable of performing VH-KI–to–DJH rearrangement in a context where endogenous VH gene segment rearrangement is prohibited, and that such rearrangement does not require Pax5. Indeed, VH-KI rearranges on a Pax5-null background in the bone marrow as well (Fig. S1). Thus, position within the IgHC locus contributes to the lineage specificity of VH-to-DJH rearrangement, and T cells possess all of the factors necessary to use VH gene segments in V(D)J recombination.
The order of recombination is not tightly regulated in VH-KI mice
In developing B cells, DH gene segments almost invariably rearrange to JH gene segments before VH-to-DJH rearrangement (4). To test whether the VH-KI gene segment undergoes normally ordered rearrangement, we assayed genomic DNA from wild-type, heterozygous, and homozygous VH-KI mice for direct VH-to-DHQ52 rearrangement over a distance of ∼60,000 nucleotides (Fig. 5 A). As expected, we failed to detect VH-to-DH rearrangements of either VH558 or VH7183 family VH gene segments in wild-type bone marrow. VH7183-to-DHQ52 rearrangements were detected sporadically and at low levels in wild-type spleen and thymus DNA samples. In contrast, we detected significant levels of such rearrangements in VH-KI DNA samples from all three tissues in the targeted mice (Fig. 5 B). These rearrangements occurred by deletion and not inversion, because the downstream primer was 3′ of the DQ52 gene segment and not within the DH gene segment itself. Thus, the ordered regulation of IgHC gene assembly is dependent on the position of VH gene segments within the locus.
The targeted VH gene segment violates allelic exclusion at the levels of rearrangement and protein expression
It has been suggested that ordered IgHC assembly might be necessary for effective allelic exclusion (3). Because we observed that positioning VH-KI proximal to DFL16.1 resulted in an increased frequency of direct VH-to-DH rearrangement, we went on to ask whether IgHC allelic exclusion was disrupted by this mutation as well. We addressed this possibility in two ways. First, we assayed genomic DNA purified from FACS-sorted wild-type and VH-KI bone marrow pro– and pre–B cells and thymocytes for dsDNA breaks at various RSSs (Fig. 6 A). Such dsDNA breaks are reaction intermediates in V(D)J recombination and indicate active rearrangement of the gene segment under study at the time of DNA isolation (27). Pro–B cells were defined as B220+CD43+ intracellular μ− (icμ−) and pre–B cells as B220+CD43−icμ+. Our analysis revealed that pro–B cells contain dsDNA breaks at RSSs 5′ of DFL16.1 but not at those 5′ of Jκ5. In contrast, wild-type pre–B cells possess abundant breaks at RSSs 5′ of Jκ5, but not at those 5′ of DFL16.1. Remarkably, in the targeted animals, RSS breaks are easily detectable in pre–B cell DNA both 5′ of DFL16.1 and 3′ of VH-KI, indicating active VH-KI–to–DH rearrangement in violation of allelic exclusion (Fig. 6 A). The primers used to amplify 3′of VH-KI signal end breaks were not degenerate and, therefore, did not amplify other members of the VH gene family. Breaks at the VH-KI endogenous counterpart in wild-type pro–B cells were below the level of detection. However, we could not discern whether VH-KI or 5′ of DFL16.1 RSS breaks occur on unrearranged or DJH-rearranged knock-in alleles. Nonetheless, we conclude that the targeted VH gene is able to undergo rearrangement into the pre–B cell stage, when endogenous VH-to-DJH recombination is silenced by allelic exclusion.
To further examine IgHC allelic exclusion, we bred a well-characterized human μ (hμ) transgene onto either a wild-type or VH-KI heterozygous genetic background. Expression of a transgenic hμ protein in developing B cells inhibits endogenous VH-to-DJH rearrangement and surface expression of mouse IgHC on splenic B cells (Fig. 6 B, continuous line) (7). In contrast, the hμ transgene has far less of an effect on the expression of mouse IgHC protein in VH-KI mice (Fig. 6 B, dashed line). Flow cytometric analysis of surface mouse IgM/IgD expression on B220+ splenocytes in hμ-transgenic mice revealed a fourfold increase in mouse μ expression when one targeted VH-KI allele is present (Fig. 6 B).
We went on to examine the frequency of V(D)J-rearranged alleles in bone marrow cDNA from wild-type and VH-KI mice in the presence of the hμ transgene. This analysis confirmed that the targeted VH gene was indeed abundantly rearranged in the bone marrow of targeted animals on the hμ transgenic background (Fig. S2). Collectively, these experiments show that repositioning a VH558 gene segment to a location proximal to DFL16.1 results in the disruption of IgHC locus allelic exclusion.
We have demonstrated that the chromosomal position of a VH gene segment within the IgHC locus rather than VH gene–associated sequences greatly influences the frequency, order, and cell-type specificity of its rearrangement. Eμ, the only known enhancer in the JH region of the locus, is required to promote optimal accessibility of the IgHC locus for both DH-to-JH and VH-to-DJH rearrangement, yet DH-to-JH rearrangement consistently precedes VH-to-DJH rearrangement (4, 21–23). Moreover, several recent studies have demonstrated that locus contraction occurs in correlation with but independently of V-to-DJ rearrangement (15–17). We hypothesized that differential regulation of the clusters of DH and VH gene segments depends on the distance between these sequences within the nucleus. Through the repositioning of a normally distal VH558 gene segment, this is precisely what we observed.
The frequency with which the VH-KI gene segment rearranges is greatly enhanced by repositioning, which was quite unexpected considering similar experiments in T cells at the TCRβ locus (28). The TCRβ locus is much like the IgHC locus in that it has V, D, and J gene segments. It is the first locus to undergo rearrangement in T cells and, thus, like the IgHC locus, requires silencing to enforce allelic exclusion during TCRα rearrangement. Vβ gene segments are separated from Dβ and Jβ gene segments by ∼350 kb of DNA, with the exception of Vβ14, which lies ∼10 kb downstream of the DJCβ clusters on the far side of the only known enhancer in the locus, Eβ (29). The TCRβ locus undergoes ordered and cell type–specific rearrangement: Dβ-to-Jβ is followed by Vβ-to-DJβ rearrangement only in T cells and is strictly dependent on Eβ. Eβ deletion results in the absence of germline transcription that normally precedes any rearrangement, and homozygous Eβ-deleted animals lack αβ T cells altogether (30, 31). The domain of chromatin structure regulated by Eβ, as identified by a restriction enzyme accessibility assay, extends only 2 kb upstream of Dβ1 (32).
Insertion of a targeted Vβ gene segment ∼7 kb upstream of Dβ1 did not increase its frequency of rearrangement (26). However, deleting Dβ1 along with the 350-kb Vβ-Dβ interval resulted in a significant increase in the frequency of rearrangement of those Vβ gene segments now positioned much closer to Eβ (33). Thus, only when the entire Vβ-to-Dβ interval was deleted did the frequency of Dβ gene segment–proximal Vβ genes segments increase. This could be explained by the presence of a boundary element in the Vβ-to-Dβ interval that prevents the spreading of open chromatin from extending to the Vβ gene segments even when they are brought much closer to the Dβ and Jβ gene segments. Similarly, it is possible that the increase in rearrangement frequency of VH-KI is caused by its position within the realm of accessibility potentially limited by an analogous chromosomal boundary.
Lineage specificity and the role of transcription factors and histone modifications in VH-to-DJH rearrangement
We found that the chromosomal context of a VH gene segment influences the lineage specificity of its rearrangement. Previous studies had shown that transgenic expression of Pax5 in thymocytes was sufficient to activate VH-to-DJH rearrangement and cause compaction of the distal and proximal regions of the IgHC locus (14, 20). More recently, it was shown that Pax5 can bind directly to a subset of VH gene segments and can recruit the recombinase to these VH gene segments via a protein–protein interaction (19). Pax5 had also been shown to be necessary for removal of inhibitory histone methylation around the distal VH gene segments (34). The 1.3 kb of 5′ of VH promoter region sequence upstream of the targeted VH gene does contain potential Pax5 binding sites, but VHKI–to–DJH rearrangement was independent of Pax5 in thymocytes and in IL-7–dependent bone marrow culture, calling into question an obligatory role for this transcription factor in IgHC V(D)J recombination (Fig. S1). Our results are more consistent with Pax5 regulating VH-to-DJH rearrangement by bringing distal VH gene segments into proximity with DH segments (compaction).
IL-7 signaling has been proposed to play a role in IgHC allelic exclusion by regulating STAT5 binding to VH gene segment promoters (18, 35, 36). This idea was recently challenged by the observation that allelic exclusion is intact in the presence of a constitutively active STAT5 (37). Our data also argue against a role for VH gene promoters in establishing allelic exclusion because the targeted VH gene promoter is not sufficient to enforce allelic exclusion. It remains possible, however, that IL-7 signaling is required for VH gene activation, because IL-7 signaling does play a role in T cell development (38, 39). Indeed, in STAT5ab−/− mice, Pax5 and Ezh2 expression are normal and chromosomal contraction occurs, but rearrangement is nonetheless impaired (36). Thus, STAT5 binding to VH gene promoters and subsequent histone acetylation may be necessary to promote but not sufficient to properly regulate VH gene segment activation. VH gene segment promoters and RSSs from RAGnull IL-7–dependent pro–B cells are H3K4 methylated, but sorted double-negative thymocyte VH gene segment promoters and RSSs remain unmethylated at H3K4, suggesting a role for this modification in the activation of VH gene segment recombination (34). In agreement with this, we see recruitment of H3K4 methylation to the targeted VH gene segment RSS in both IL-7–dependent bone marrow culture from RAGnull heterozygous VH-KI mice and primary thymocytes from RAGwt heterozygous VH-KI mice.
Various mechanisms have been proposed to explain the ordered nature of IgHC gene assembly. One such mechanism involves the preferential binding of RAG complexes to 3′ of DH RSSs, limiting the accessibility of the RAGs to the 5′ of DH RSSs until after DH-to-JH rearrangement deletes the 3′ of DH RSS. This, however, cannot be the case, because we see direct VH to DH joining on the targeted locus. It is worth noting that we only observed direct VH-KI–to–DH rearrangements involving the DQ52 gene segment; no direct VH-KI–to–DSP2 family gene segment rearrangement was observed (unpublished data). Promoters upstream of the DH gene segments become active upon DH-to-JH rearrangement (40, 41), and it may be that rearrangement-induced transcription attracts VH genes to rearranged DJH gene segments. Although not all DH gene segments were tested, the observation that DQ52 is noticeably available for direct VH-KI–to–DH rearrangement could be a reflection of the promoter upstream of DQ52 (driving the μ° germline transcript), which is active before DH-to-JH rearrangement (40, 42). The failure of VH genes in their normal chromosomal positions to rearrange to the accessible DQ52 might be caused by boundary element activity or simply distance within the nucleus.
Allelic exclusion and VH gene segment position
Rearrangement of the targeted VH-KI allele was not subject to allelic exclusion imposed by an hμ transgene. This observation is consistent with previously published results showing that endogenous DH-proximal VH gene segments continue to rearrange at a low but detectable frequency in IgHC transgenic mice (8). What is surprising is that the targeted VH-KI gene segment was frequently expressed, increasing the fraction of cells concomitantly expressing both mouse and human IgHC by fourfold compared with wild-type hμ transgenic mice. This is in contrast to the inserted Vβ gene segment within the TCRβ locus, where in the presence of a transgenic TCRβ the targeted gene was not subject to allelic exclusion at the level of rearrangement, but protein expression was inhibited (28).
The targeted VH-KI gene segment also escaped allelic exclusion in nontransgenic mice. We detected dsDNA RSS breaks 3′ of VH-KI as well as 5′ of DFL16.1 in sorted pre–B cell DNA from heterozygous knock-in but not wild-type animals. We detected a much stronger dsDNA RSS break signal from the targeted VH-KI gene segment as compared with endogenous VH gene segments in the pro–B cell samples, which can be explained by the increased frequency of rearrangement of the targeted gene segment (Fig. 2). The persistence of 5′ of DFL16.1 breaks in pre–B cells and thymocytes from VH-KI knock-in but not wild-type mice speaks more accurately to the contrast between the wild-type and heterozygous animals. Thus, targeting a VH gene segment to this chromosomal position affects the inactivation as well as the activation of the VH gene segment rearrangement.
The chromosomal domain model
Changes in the proximity of VH gene segments to Eμ upon DH-to-JH rearrangement does not adequately explain the order, frequency, and cell-type specificity of VH gene segment rearrangement in wild-type mice. DQ52 rearrangement to JH gene segments deletes as little as 700 bp of DNA, hardly enough to significantly alter the configuration of a 2-mb locus. Additionally, the targeted VH-KI gene segment can rearrange directly to DQ52, which is over 80 kb away, roughly the same length of DNA that normally separates VH81X and DFL16.1 (∼90 kb). This argues that distance alone might not account for such radical changes in the regulation of VH gene segment recombination. We have demonstrated that DH-to-JH rearrangement itself does not account for the activation of the VH-KI gene segment, because direct VH-KI–to–DH rearrangements are observed. It is possible that the region 5′ of DFL16.1 into which we inserted VH-KI has enhancer or promoter activity or that the region of H3K4 methylation 5′ of DFL16.1 in B cells (Fig. 3, A and B) (26) stimulates V(D)J recombination, but this cannot account for the activation of VH-KI gene segment rearrangement in thymocytes. Of the two histone modifications we explored, the only modifications seen in thymocytes at this locus are those recruited by the targeted VH gene, not by the endogenous locus (Fig. 3).
What might explain readily detectable VH-KI rearrangement in thymocytes? We favor the possibility that VH and DH gene segments normally occupy distinct chromosomal domains, but the targeted VH-KI gene segment now lies within the DH domain. This DH domain is normally accessible to the recombinase in both developing B and T cells. Our results lead us to two possible models. The VH genes themselves can undergo activation in either B or T cells, but the timing, frequency, and lineage specificity of VH-to-DJH rearrangement may be dependent on either (a) locus compaction or (b) a VH domain control element that is unable to affect the repositioned VH gene. Both of these models require that the VH and DH domains remain functionally separate, suggesting the existence of a boundary element. We are in the process of targeting the VH-KI gene segment to a series of locations progressively further from DFL16.1.
Materials And Methods
63-12 (RAG2null Abelson murine leukemia virus [AMuLV]–transformed pro–B cell ; provided by F. Alt, Harvard Medical School, Boston, MA), P5424 (RAG2null, p53null pro–T cell ; provided by P. Mombaerts, The Rockefeller University, New York, NY), and Pax5null (AMuLV-transformed pro–B cell line derived from mice containing LacZ in place of exon 2 of Pax5 ; provided by M. Busslinger, Research Institute of Molecular Pathology, Vienna, Austria) cell lines were grown at 37°C/5% CO2 in RPMI 1640 supplemented with 5–10% FCS, antibiotics, and 50 μM β-mercaptoethanol.
Primary pro–B cell culture.
Bone marrow was harvested from the femurs of 4–8-wk-old mice and rid of red blood cells by ACK lysis. Cells were cultured on S17 stromal cells in the presence of 10% IL-7–containing culture supernatant in RPMI 1640 with 10% FCS, antibiotics and 50 μM β-mercaptoethanol. After culture, >95% of cells stained positively for surface CD19 or B220. Primary thymocytes were purified by centrifugation with HISTOPAQUE-1083 (Sigma-Aldrich), after which they were >90% CD4/CD8 positive. Animal experimentation procedures were approved by the University of California, Berkeley Animal Care and Use Committee.
ChIP was performed as previously described (46). 10 μg of antidimethyl H3K4 antibody (Millipore), 15 μl of antisera against acetylated histone H3 (Millipore), or 10 μg of normal rabbit IgG (Santa Cruz Biotechnology, Inc.) were used for IP. The ratio of immunoprecipitated to input DNA for a given genomic region (IP/input) was defined as 2^(Ctinput-CtIP), where Ct is the cycle threshold for real-time PCR with SYBR green technology. Chromatin from cell lines was precleared with protein A/G–sepharose (Millipore) blocked with sheared salmon sperm DNA and BSA, whereas chromatin from primary cells was precleared with unblocked sepharose before IP. Error bars represent one SD from an average of two experiments. 5 × 106 cell equivalents were used per IP for cell lines, and 2.5 × 106 cell equivalents were used for primary cells. PCR primers are listed in Supplemental materials and methods .
Targeting construct and VH-KI mutant mice.
The targeting vector consisted of a left arm of 5.6 kb (from 74623–80260 of the bacterial artificial chromosome [BAC] available from GenBank/EMBL/DDBJ under accession no. AC073553), the VH gene segment in the sense orientation, a loxP-flanked neomycin resistance cassette in the opposite transcriptional direction, and a right arm of 2.5 kb (from 80260–82746 of the same BAC) of homologous sequence. The VH gene was targeted to a position ∼500 bp 5′ of DFL16.1 (∼80700 of the same BAC) in the same transcriptional orientation as DFL16.1. Cloning of the VH gene segment and probes used for Southern blot analyses are described in Supplemental materials and methods.
Cell staining and sorting.
Spleen and thymus were strained through a 40-μm cell strainer, whereas femurs and tibias were dissected from mice and crushed with a mortar and pestle or flushed with a syringe. Lymphocytes were isolated by density centrifugation using HISTOPAQUE-1083. Antibodies used for sorting DP thymocytes were anti-CD8α–FITC (BD Biosciences) and CD4-PE (BD Biosciences). For transgenic hμ analyses, splenocytes were stained with B220-PE (BD Biosciences), IgM-biotin (clone II/41; BD Biosciences), and IgD-biotin (clone 11–26; eBioscience) with streptavidin-Cy5 (BD Biosciences). Antibodies used for sorting cells for ligation-mediated PCR (LM-PCR) were rat anti–mouse IgM-biotin (clone 1B4B1; SouthernBiotech), IgD-biotin (for magnetic bead depletion; SouthernBiotech), CD43-biotin (clone S7; BD Biosciences) with streptavidin-cychrome (BD Biosciences), B220-PE (BD Biosciences), and anti–mouse IgM-FITC (clone II/41; BD Biosciences), for intracellular staining after fixation in 1% paraformaldehyde and permeabilization with 0.1% saponin.
Online supplemental material.
Fig. S1 shows that Pax5 is not necessary for VH-KI rearrangement in developing B cells. Fig. S2 shows that VH-KI is not subject to allelic exclusion. Supplemental materials and methods provides information about targeting construct cloning and probes for Southern screening, as well as primers used for PCR assays, including ChIP, frequency of VH-KI gene rearrangement, recombination assays, and LM-PCR.
We thank M. Busslinger for Pax5null mice, P. Mombaerts for the P5424 pro–T cell line, and F. Alt for the 63-12 pro–B cell line.
M.S. Schlissel acknowledges support from the National Institutes of Health (grants RO1 HL48702 and AI40227).
The authors have no conflicting financial interests.
Abbreviations used: BAC, bacterial artificial chromosome; cDNA, complementary DNA; ChIP, chromatin immunoprecipitation; DP, double positive; dsDNA, double-stranded DNA; hμ, human μ; icμ, intracellular μ; LM-PCR, ligation-mediated PCR; RSS, recombination signal sequence.
J.G. Bates's present address is Stanford University, Stanford, CA 94305.