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. 2007 Sep 6;26(19):4273–4282. doi: 10.1038/sj.emboj.7601846

Transcription of productive and nonproductive VDJ-recombined alleles after IgH allelic exclusion

Janssen Daly 1,*, Steve Licence 1, Aikaterini Nanou 2, Geoff Morgan 3, Inga-Lill Mårtensson 1,a
PMCID: PMC2230841  PMID: 17805345

Abstract

The process of allelic exclusion ensures that each B cell expresses a B-cell receptor encoded by only one of its Ig heavy (IgH) and light (IgL) chain alleles. Although its precise mechanism is unknown, recruitment of the nonfunctional IgH allele to centromeric heterochromatin correlates with the establishment of allelic exclusion. Similarly, recruitment in activated splenic B cells correlates with cell division. In the latter, the recruited IgH allele was reported to be transcriptionally silent. However, it is not known whether monoallelic recruitment during establishment of allelic exclusion correlates with transcriptional silencing. To investigate this, we assessed the transcriptional status of both IgH alleles in single primary cells over the course of B-cell development, using RNA fluorescence in situ hybridization. Before allelic exclusion both alleles are transcribed. Thereafter, in pre-BII and subsequent developmental stages both functional and nonfunctional VDJ- and DJ-transcription is observed. Thus, after the establishment of IgH allelic exclusion, monoallelic recruitment to heterochromatin does not silence VDJ- or DJ-transcription, but serves another purpose.

Keywords: allelic exclusion, B-cell development, heterochromatin, IgH transcription

Introduction

B-cell development results in the production of naïve B cells, each expressing a single, clonotypic B-cell receptor (BCR) on their cell surface (Rajewsky, 1996). The primary diversity of the BCR, composed of Ig heavy (HC) and light (LC) chains, is guaranteed by V(D)J recombination of the respective loci, whereas allelic exclusion ensures that the BCR is encoded by only one IgH and one IgL allele. During early B-cell development, V(D)J recombination takes place in a stepwise fashion: first DH to JH recombination, which occurs on both alleles in the majority of cells. According to the regulated model of allelic exclusion (Alt et al, 1980, 1984; Jung et al, 2006), this is followed by VH to DJ-recombination on one of the two alleles. If productive, this leads to the expression of a μHC, which mediates a feedback signal that inhibits further recombination of the second, DJ, allele. However, if nonproductive, the second allele undergoes VH to DJ-recombination and if this is productive, the cell expresses a μHC and is able to progress further in development, whereas if nonproductive, the cell dies. In addition, expression of a functional μHC results in downregulation of the recombination activating genes (RAG). Thereafter, the cells undergo a burst of proliferation, driven by the pre-B-cell receptor (pre-BCR), that is, μHC in association with surrogate LC (Melchers, 2005; Martensson et al, 2007). As the cells leave the cell cycle, the RAG genes are reactivated and the IgL (κ/λ) loci undergo VJL recombination. Following its expression, LC assembles with μHC to form a cell-surface-bound BCR, which mediates LC allelic and isotype exclusion and downregulation of RAG gene expression.

Although the precise mechanisms of VDJ-recombination and IgH allelic exclusion remain unknown, in recent years several processes have been described that may contribute (Bergman and Cedar, 2004; Corcoran, 2005; Geier and Schlissel, 2006; Jung et al, 2006; Sen and Oltz, 2006; Spicuglia et al, 2006; Fuxa and Skok, 2007). The IgH alleles are repositioned away from the nuclear periphery to the center of the nucleus, the locus undergoes looping and contraction followed by decontraction and, in addition, sense and antisense transcription takes place in the VH region, all of which are lineage- and stage-specific events. Moreover, in pre-B but not in pro-B cells, the nonfunctional, that is, DJ or nonproductive VDJ allele is recruited to centromeric heterochromatin (Roldan et al, 2005), thus correlating with IgH allelic exclusion. Similarly, in activated but not resting splenic B cells, monoallelic recruitment to centromeric heterochromatin takes place (Skok et al, 2001), thus correlating with cell division (Brown et al, 1999). In addition, in activated splenic B cells, the recruited allele was found to be transcriptionally silenced (Skok et al, 2001). This has led to a model where monoallelic recruitment to centromeric heterochromatin ensures maintenance of IgH allelic exclusion during cell division (Fisher, 2002). The transcriptional status of both IgH alleles in single primary cells over the course of early B-cell development has not been investigated in detail. Thus, it is not known whether monoallelic recruitment to heterochromatin in pre-B cells, that is, establishment of IgH allelic exclusion, correlates with transcriptional silencing of the recruited IgH allele. Therefore, we set out to investigate the transcription pattern of both IgH alleles at sequential stages of B-cell development, in sorted primary BM and spleen cells, using RNA FISH. We demonstrate that both VDJ- and DJ-transcription takes place after IgH allelic exclusion. This implies that recruitment to centromeric heterochromatin does not silence VDJ- or DJ-transcription on the nonfunctional IgH allele, but rather plays a role in some as yet unknown mechanism.

Results

VDJ- and DJ-transcription in allelically excluded B-lineage cells

To investigate the transcriptional status of the IgH locus during B-cell development, the following cell populations were analyzed (Figure 1A). From RAG1-deficient (RAG−/−) mice (Spanopoulou et al, 1994): CD19+ bone marrow (BM) cells, with both IgH alleles in GL configuration due to their inability to recombine their Ig loci (Mombaerts et al, 1992; Shinkai et al, 1992; Spanopoulou et al, 1994). From wt (C57BL/6) mice: BM pre-BI cells, most of which represent a population before IgH allelic exclusion (∼75% are DJ-recombined); BM pre-BII and immature/mature B cells in addition to splenic B cells before and after activation with LPS and IL-4, all of which represent allelically excluded cell populations. Based on single-cell PCR, ∼50% of a population of allelically excluded cells are VDJ/DJ and the remaining VDJ/VDJ-recombined (Ehlich et al, 1994; ten Boekel et al, 1995), where one allele is functional and the other nonfunctional, either DJ or nonproductive VDJ.

Figure 1.

Figure 1

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RT–PCR analysis of steady-state IgH RNA levels during normal B-cell development. (A) Schematic representation of BM B-cell development, indicating the recombination status of the IgH and IgL loci and expression of the pre-BCR and BCR. (B) Schematic representation (not to scale) of the IgH locus, germline (GL), DJ- or VDJ-recombined together with the relative positions of the primers used for RT–PCR. (C) Semiquantitative RT–PCR for the indicated transcripts using RNA prepared from (top) BM pre-BI, splenic B cells enriched by CD43-depletion (d0) or after activation with LPS and IL-4 for 2 and 4 days (d2 and d4, respectively) and ST-2 stromal cells and (bottom) BM pre-BI and pre-BII cells.

First, we investigated representative cell populations by RT–PCR, to determine mRNA steady-state levels on a population basis. Previous studies have defined several types of IgH transcription initiated at various promoters: μo, driven by the DQ52 promoter; Iμ, initiated at the Iμ promoter as well as DJ- and VDJ-transcription driven by the D and VH promoters, respectively (Lennon and Perry, 1990). Here, we used a 5′ primer recognizing either VJ558 (VJ558 is used in 40–80% of functional VDJ-alleles), DSP/DFL (most frequently recombined D gene segments; Bangs et al, 1991) or Iμ, together with a 3′ primer in Cμ1 to detect VDJ-, DJ- and Iμ-transcription, respectively (Figure 1B). Additional primers were used to detect Cμ and Cγ1. In this assay, VJ558DJ-, DJ-, Iμ- and Cμ-mRNA was detected in all cell populations analyzed, whereas Cγ1-mRNA was only detected in splenic B cells activated with LPS and IL-4 (Figure 1C and data not shown). The detection of VJ558DJ-mRNA in all cell populations analyzed was expected, as all express a μHC (∼20% of pre-BI cells are μHC+; Martensson et al, 2002). Furthermore, the presence of DJ-transcripts in pre-BI cells is consistent with the majority of these being DJ-recombined. However, that the nonfunctional, DJ-allele, gave rise to DJ-transcription in pre-BII as well as resting and activated splenic B cells, that is in cells that have undergone IgH allelic exclusion, was unexpected. Although the frequency of cells in which the DJ-allele is active cannot be deduced from these results, the RT–PCR data, nonetheless, suggest that transcription occurs on a large proportion of DJ-alleles; the signal was not reduced in pre-BII or late B compared to pre-BI cells, even though only one allele in each cell is DJ-recombined and this in only half the population. Therefore, this observation suggests that establishment/maintenance of IgH allelic exclusion, does not result in transcriptional silencing of the DJ-allele. That the DJ-allele is active in late B cells contrasts with previous studies, that recruitment to centromeric heterochromatin results in silencing of the nonfunctional IgH allele in activated splenic B cells (Skok et al, 2001).

Mono- and biallelic IgH transcription after IgH allelic exclusion

To clarify the above results, an allele-specific assay was required to determine whether both the functional and nonfunctional IgH allele is active. We used RNA FISH, as this technique would enable us to distinguish primary transcription on individual alleles in single cells. To detect transcription at the IgH locus, an Iμ (Figure 2A) and a Cμ (data not shown) probe were used in combination with a CD45 probe (Skok et al, 2001), that is B220, which is expressed in all cell populations and is biallelically transcribed (Parker et al, 2005).

Figure 2.

Figure 2

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RNA FISH analysis of IgH transcription during normal B-cell development. (A) Schematic representation (not to scale) of the IgH locus, together with the relative positions of the probes used for FISH analysis and VDJ-recombination. (B) Composite images of: wt pre-BI (i) and pre-BII (ii, iii) cells analyzed by RNA FISH using Iμ (red) combined with (i, ii) CD45 (green) or (iii) VJ558 (green); RAG−/− CD19+ BM cells analyzed by DNA FISH using (iv) CR (green) or (v) IR1 (green); wt splenic B cells analyzed by combined RNA/DNA FISH using Iμ (red) combined with (vi) CR (green) or (vii) IR1 (green). DAPI (blue) allows visualization of nuclear DNA. (C) The proportion of nuclei with signals. (D) The mean±s.e. of signal-positive nuclei with mono- and biallelic transcription, using the CD45 (C) combined with the Iμ (Iμ) probe. (E) The proportion of transcriptionally active IgH alleles in signal-positive nuclei. Cell populations: CD19+ BM cells from RAG−/− mice; BM pre-BI, pre-BII and B cells from wt mice; splenic B (Spl B) cells from wt mice after CD19-enrichment (similar results were obtained with B220+IgM+-sorted cells, not shown), or enriched by CD43-depletion (day 0) followed by activation with LPS and IL-4 (days 2 and 4). In each experiment, >100 nuclei were counted per slide. Number of nuclei analyzed: 243 (RAG−/−), 779 (pre-BI), 1164 (pre-BII), 1209 (BM B), 1317 (Spl B), 374 (Day 0), 145 (Day 2), 453 (Day 4).

Hybridization with both the CD45 and Iμ probes resulted in discrete foci in the nuclei of all cell populations analyzed (Figure 2B). Both the CD45 and Iμ probe gave rise to a high (⩾70%) proportion of nuclei with signals (Figure 2C). In all cell populations and in nuclei with CD45 signals, approximately 95% of alleles were transcriptionally active (data not shown); the majority (90%) of nuclei contained two foci (Figure 2D). Thus, as expected, the CD45 gene is predominantly biallelically transcribed from the early to late stages of B-cell development.

Analyses using the Iμ probe demonstrated that, while 90–95% of alleles in CD19+ BM cells from RAG−/− and the pre-BI population of wt mice were active, in those cell populations that had undergone IgH allelic exclusion it is ∼80%. (Figure 2E). Although this suggested that the majority of alleles were active, there was nevertheless a difference between cells before and after IgH allelic exclusion, which was therefore analyzed in more detail. In CD19+ BM cells from RAG−/− mice, the majority (∼85%) of nuclei with Iμ signals contained two foci (Figure 2D), that is a pattern similar to that of CD45, suggesting that in cells in which both IgH alleles are in GL configuration, the predominant transcription pattern is biallelic. Similarly, 85% of nuclei from the pre-BI population of wt mice also contained two Iμ foci and, as the majority of pre-BI cells are DJ/DJ-recombined, this suggests that transcription takes place on both DJ alleles. In contrast, a significant change was observed in pre-BII cells, in which the proportion of nuclei with one Iμ signal increased to ∼40% (P=0.001) and those with two decreased to ∼60% (P>0.001) (Figure 2D). A similar transcription pattern was observed at all subsequent stages of B-cell development, including activated splenic B cells. The results from BM pre-BI, pre-BII, B and splenic B cells were confirmed using the Cμ probe (data not shown). This demonstrates, therefore, that from the pre-BII stage and onwards, the proportion of nuclei with one Iμ signal remained constant at ∼40% and those with two Iμ signals made up the remaining 60%. Thus, by RNA FISH, biallelic IgH transcription is detected before IgH allelic exclusion whilst after, some cells transcribe both and others only one IgH allele.

Mono- and biallelic transcription originate from VDJ-recombined alleles

Both the RT–PCR, which detects mRNA steady-state levels, and the RNA FISH, which detects primary transcription, analyses suggested that both IgH alleles are transcribed before IgH allelic exclusion. However, in allelically excluded cells, given that the RT–PCR results suggested that both VDJ- and DJ-transcription was taking place, the source of transcription detected using RNA FISH was unclear as both the Iμ and Cμ probes can detect μo-, Iμ-, DJ- and VDJ-transcription. Therefore, we asked whether the transcription observed in allelically excluded cells represented VDJ transcription by combining the Iμ probe with a VH probe in a RNA FISH assay (Figure 2A). A VHJ558 probe (Bolland et al, 2004) was used as this family is the most frequently recombined and found on both productive and nonproductive VDJ-recombined alleles, with an overall usage of ∼60% (ten Boekel et al, 1997). In those cells which have recombined a VJ558 family member and if VDJ-transcription is taking place, then the Iμ and VJ558 probe signals should colocalize. In this analysis, the VJ558 signals, either one or two, were only detected in nuclei with Iμ signals, where they colocalized (Figure 2B). In BM pre-BII cells, whereas 21% of nuclei contained only one Iμ signal, 14% contained one Iμ colocalized with one VJ558, 33% contained two Iμ foci, while two Iμ and one VJ558 were found in 20% and two of each in 12% (Table I). Similar results were obtained from both BM and splenic B cells (Table I; data not shown). Thus, VJ558 signals were observed in Iμ-positive nuclei from BM pre-BII, B and splenic B cells with an overall usage of 45–60%, comparable to the previously reported frequency (∼60%) of VHJ558 recombination. Furthermore, in a proportion of these nuclei, the two Iμ signals were found to colocalize with two VJ558 foci, suggesting that the nonproductively recombined VDJ-cassette is also transcribed. Thus, these data demonstrate that VDJ-transcription contributes to both the mono- and biallelic pattern observed in IgH allelically excluded cells.

Table 1.

VDJH-transcription assay

Cell population VJ558 usagea Iμ:VJ558b
    1:0 1:1 2:0 2:1 2:2
Pre-BII 46 21 14 33 20 12
Spl B
 45 20 17 35 22 6
Nuclei from the indicated cell populations were analyzed by RNA FISH using the Iμ combined with the VJ558 probe.
Number of nuclei: 113 (pre-BII) and 110 (splenic B).
aVJ558 usage as a proportion of nuclei with Iμ signals.
bPercentage of nuclei with either one or two Iμ foci together with 0, 1 or 2 VJ558 signals.
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Ratio of VDJ/VDJ- and VDJ/DJ-recombined B cells

The ratio of nuclei with one and two Iμ foci (40:60), observed in IgH allelically excluded cells, is similar to the reported ratio (∼50:50) of VDJ/DJ- and VDJ/VDJ-recombined cells, determined by single-cell PCR (Ehlich et al, 1994; ten Boekel et al, 1995; Rajewsky, 1996; Melchers et al, 1999). A possible explanation for our RNA FISH results using the Iμ and VJ558 probes may be, therefore, that monoallelic transcription originates from VDJ/DJ-recombined cells while those with two signals represent VDJ/VDJ-recombined cells. Given that the detection level in single-cell PCR is relatively low (15–40%) (Ehlich et al, 1994; ten Boekel et al, 1995), we determined the proportion of VDJ/VDJ recombined cells using DNA FISH. For these experiments, a probe (IR1) was used that recognizes the intergenic region between the V and D gene segments, that is, hybridizes to GL and DJ- but not VDJ-recombined alleles, whereas the constant region (Cδ-Cα) probe (CR) recognizes the IgH allele regardless of its recombination status (Figure 2A and B). In CD19+ BM cells from RAG−/− mice, with both IgH alleles in GL configuration, signals were observed in ∼80% of nuclei using either probe and, of those, two foci were observed in >85% (Supplementary Figure 1A). Thus, the detection level was high and comparable for both probes, with the CR probe reflecting detection levels and the presence of one IR1 signal indicating VDJ/DJ- and the absence of IR1 foci VDJ/VDJ-recombined cells.

In wt pre-BII and splenic B cells, the detection level (CR probe) was similar, 77 and 76%, respectively, to that observed in CD19+ BM cells from RAG−/− mice, while the proportion of nuclei with IR1 signals was reduced; 42 and 35%, respectively (Supplementary Figure 1B). From this, the percentage of VDJ/VDJ-recombined cells was calculated to be 46% in pre-BII and 54% in splenic B cells (Supplementary Figure 1C). These results are supported by the observation that most of the remaining nuclei contained one IR1 signal, in agreement with one allele being DJ-recombined and the other VDJ. Because the majority of splenic B cells are resting, together with the high sort purities (95–99%), these results demonstrate that in a population of allelically excluded cells, 54% are VDJ/VDJ-recombined and the remaining VDJ/DJ-recombined, which is similar to the proportions previously obtained using single-cell PCR.

Both productive and nonproductive VDJ alleles are transcriptionally active

To investigate more directly whether one Iμ probe transcription signal stems from VDJ/DJ-recombined cells and those with two from VDJ/VDJ, a combined RNA/DNA FISH assay was performed on splenic B (Figure 3) and pre-BII (data not shown) cells using the Iμ probe in combination with either the CR or IR1 probe (Figure 2A and B). In this combined assay, the detection level for these probes was similar to that in previous experiments (data not shown). The control probe (CR) gave rise to two foci in the majority (85–90%) of cells and the ratio of one versus two Iμ signals was ∼40:60 (Figure 3). In the majority (∼90%) of nuclei with one Iμ signal, the IR1 probe gave rise to one signal; the two being well separated (Figures 2B and 3). The majority (∼85%) of nuclei with two Iμ signals lacked IR1 probe foci. These data demonstrate that the majority of nuclei with one Iμ signal are VDJ/DJ-recombined and transcribe the VDJ-allele, whereas those with two Iμ signals represent VDJ/VDJ-recombined cells. Thus, after IgH allelic exclusion, VDJ-recombined alleles, whether productive or nonproductive, are transcribed.

Figure 3.

Figure 3

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VDJ-recombined alleles are transcriptionally active after IgH allelic exclusion. Combined RNA/DNA FISH analysis using the Iμ probe combined with either the CR or IR1 probe. The ratio (43:57) of one to two Iμ probe signals in splenic B cells using the CR probe is shown in the central histogram (40:60 using the IR probe, data not shown) and the proportion of these with either 0, 1 or 2 CR (left) and either 0, 1 or 2 IR1 (right) foci within nuclei containing one or two Iμ signals. Number of nuclei analyzed: 100 (IR1) and 86 (CR).

Increase in biallelic transcription in splenic B cells from μMT+/− mice

To further substantiate the above results, we reasoned that in a population with only VDJ/VDJ-recombined B cells, that is, under conditions of allelic inclusion, transcription from both alleles should be observed in all cells. An example of this is found in the μMT mouse line, in which IgH allelic exclusion is abolished (Kitamura and Rajewsky, 1992). However, as B-cell development is blocked at the pre-BI cell stage in these mice (Kitamura et al, 1991), we used heterozygous (μMT+/−) mice in which splenic B cells develop that express surface μHCs encoded by the wt allele, while a proportion (∼6%) of cells have been shown to express HCs encoded by both IgH alleles (Kitamura and Rajewsky, 1992).

In the RNA FISH assay, the detection levels for the CD45, Iμ and Cμ probes was similar in μMT+/− and wt splenic B cells (Figure 4A and data not shown) and the CD45 gene was also predominantly biallelically transcribed in μMT+/− B cells (Figure 4B). In μMT+/− compared to wt splenic B cells, the proportion of nuclei with two Iμ or Cμ signals was significantly (P=0.01) increased to 71% and those with one, significantly (P>0.001) reduced to 29% (Figure 4B and data not shown). This represents an 11% increase in biallelically transcribing cells, which correlates well with our observed increase (12%) in VDJ/VDJ-recombined cells (Supplementary Figure 2). Although this frequency is higher than that observed at the protein level, this is to be expected, as some of the VDJ-recombinations on the targeted allele would be nonproductive. Furthermore, in the combined RNA/DNA FISH assay, the ratio of one to two Iμ signals was 29:71(±2) and in nuclei with one Iμ signal, the majority (∼90%) contained one IR1 probe signal, whereas of those with two Iμ foci, ∼90% lacked IR1 signals (data not shown). Thus, these data demonstrate that an increase in the proportion of VDJ/VDJ-recombined cells results in an increase in biallelic IgH transcription supporting our conclusion that VDJ-transcription takes place on VDJ-recombined alleles, whether functional or non functional.

Figure 4.

Figure 4

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Increase in biallelic transcription in splenic B cells from μMT+/− mice. CD19+ splenic B cells from μMT+/− mice were analyzed by RNA FISH. (A) The graph shows the detection level using the two probes compared with wt splenic B cells. (B) The histograms show the mean±s.e. of nuclei with bi- and monoallelic transcription, as determined using the CD45 and Iμ probes (similar results were obtained using the Cμ probe, data not shown). Number of nuclei analyzed: μMT+/−, 684; wt, 365.

VDJ-transcription also takes place in nonproductively VDJ-recombined cells

Although transcription takes place on both productive- and nonproductive VDJ-recombined alleles in allelically excluded cells, it was still possible that in some of these, the RNA FISH signal was due to Iμ- rather than VDJ-transcription. To eliminate this possibility, a probe (μ) located upstream of the (most 5′) Iμ start site (Lennon and Perry, 1985), was used (Figure 2A), which does not detect Iμ- but should recognize μo, DJ- and VDJ-transcription.

The detection level with the μ probe was similar to that of the Iμ (data not shown). In RAG−/− CD19+ BM and wt pre-BI cells, the proportion of signal-positive nuclei with two foci was 89 and 80%, respectively (Figure 5). In both BM and splenic B cells, the probe should detect DJ- and VDJ-transcription exclusively since GL alleles are rare and thus μo is absent. In these cells, the ratio of one versus two signals was 46:54 and therefore similar to that of the Iμ probe (40:60) and the ratio (46:54) of VDJ/DJ- to VDJ/VDJ-recombined cells. Thus, these results support the conclusion that VDJ-transcription takes place on both productively and nonproductively recombined VDJ-alleles in allelically excluded cells.

Figure 5.

Figure 5

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VDJ- and DJ-transcription. The histograms show the mean±s.e. of signal-positive nuclei with mono- and biallelic transcription, using the μ probe. Cell populations were as follows: CD19+ BM cells from RAG−/− mice; BM pre-BI, BM B and spenic B cells from wt mice. Data from BM B and splenic B cells were combined (IgM+).

DJ-transcription in cell lines

In the RNA FISH assay, DJ-transcription was detected in wt pre-BI but not in the subsequent developmental cell stages, even though these cells make DJ mRNA (Figure 1 and data not shown). The reason for this discrepancy is unclear, although one possible explanation is that transcription is low, supported by the weak signals in pre-BI cells (Figure 2B). Nonetheless, to investigate whether we could detect biallelic transcription in VDJ/DJ-recombined cells, a monoclonal cell population was required. Therefore, we investigated two pre-B cell lines, 38C-13 and 70Z/3 (Nelson et al, 1983; Lennon and Perry, 1985, 1989), which are VS107DJ/DJ- and VJ558DJ/DJ-recombined, respectively, and the VDJ-allele is functional (Supplementary Figure 3 and data not shown). In addition, the IgL locus is recombined but not expressed in the 70Z/3 cell line. As these cells express VDJ-, DJ- and Iμ transcripts on a population basis, the Iμ probe would serve to determine whether both alleles are detected in all cells.

Using the Iμ probe, we found that in both lines some cells transcribed both and others only one allele; in 38C-13 cells, the ratio of two versus one signal was 72:28 and in 70Z/3 cells 62:38 (Figure 6), with a high (>75%) detection level in both lines (data not shown). In the 38C-13 cell line, the CD45 gene was biallelically transcribed; 90–95% of alleles active (data not shown). Even though both IgH alleles are transcriptionally active in 60–70% of cells in a monoclonal cell population, some cells express only one, probably that of the functional VDJ. This suggests that one allele, presumably the DJ, alternates between ‘on' and ‘off' transcription states. Therefore, it is possible that the inability to detect DJ-transcription by RNA FISH in normal, allelically excluded cells is due to a low ‘on' frequency, as previously observed for other genes (Kimura et al, 2002; Levsky et al, 2002; Osborne et al, 2004).

Figure 6.

Figure 6

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DJ-transcription in cell lines. The histograms show the mean±s.d. of signal-positive nuclei with mono- and biallelic transcription, using the Iμ probe in the 38C-13 and 70Z/3 cell lines. For each line, a total of ∼400 nuclei were counted, with 100–150 per slide.

Recruitment to centromeric heterochromatin

Although ‘on' and ‘off' transcription states could be one explanation for the inability to detect DJ-transcription using RNA FISH, another explanation could be recruitment to centromeric heterochromatin. This is not observed in the 70Z/3 and 38C-13 cell lines (Skok et al, 2001) and, as these are immortalized, might not behave as normal VDJ/DJ-recombined cells. Previous work has shown that one IgH allele is recruited to centromeric heterochromatin in ∼70% of pre-BII cells (Roldan et al, 2005). As the allele recruited may be the nonfunctional, we investigated whether the DJ-allele was preferentially recruited to centromeric heterochromatin. This was investigated using a DNA FISH protocol that preserves nuclear integrity (Brown, 2002), with a γ-satellite probe recognizing centromeric heterochromatin (Parker et al, 2005), in combination with either the CR or IR2 probe. In pre-BII cells, the latter detects DJ but not VDJ alleles (Figure 2A). We scored recruitment to centromeric heterochromatin as follows: when there was apparent contact between the CR or IR2 probe and the γ-satellite probe signals, these were scored as ‘on', if they were relatively close together, they were scored as ‘close' and if they were clearly separated, they were scored as ‘off' (Figure 7A).

Figure 7.

Figure 7

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Recruitment to centromeric heterochromatin. DNA FISH analysis of BM pre-BII cells by confocal microscopy. (A) Confocal images of nuclei combining the IR2 (green) with the γ-satellite probe (red) demonstrating association between the DJ-allele and γ-satellite DNA scored as ‘on', ‘close' or ‘off'. Scoring: on, one allele in contact; close, one allele in close contact; off, one allele not associated. (B) Percentages of nuclei scored as on, close or no association between the IgH allele and γ-satellite DNA in pre-BII cells. The CR probe detected two alleles in the vast majority of nuclei, <5% of nuclei showing association of both signals. The IR2 probe detected one signal in 51% of nuclei. Number of signal-positive nuclei scored: CR, 220; IR2, 110.

Using the CR probe, we did not detect association between two probe foci and the γ-satellite probe in splenic B cells (79 nuclei). However, one signal was scored as ‘on' in about 5% of nuclei and 18%'close' (data not shown). Thus, in 23% of splenic B cells one IgH allele is associated with centromeric heterochromatin, a proportion similar to that previously reported, 28% (Roldan et al, 2005). In pre-BII cells, two CR probe signals were detected in the vast majority (>95%) of pre-BII nuclei and one IR2 probe signal was observed in 51%, the latter confirming that around half of this population is VDJ/DJ (data not shown). Both CR probe signals were associated with the γ-satellite probe in less than 5% of nuclei (data not shown), whereas the proportion of pre-BII nuclei with one CR probe ‘on' was ∼20% and including those that where ‘close' resulted in ∼50% scored as associated (Figure 7B). Thus, one IgH allele is recruited to centromeric heterochromatin in around half of pre-BII cells. In nuclei with IR2 probe signal, that is, VDJ/DJ-recombined cells, the proportions scored as ‘on' and ‘close' were each ∼20%, that is, association was observed in ∼40% of nuclei. Using these criteria to determine recruitment to centromeric heterochromatin, these data suggest that one allele is recruited in about half the pre-BII cell population and in ∼40% of these the DJ-allele is recruited. Provided that association with heterochromatin results in transcriptional silencing, we would expect that DJ-transcription should be detectable in the ∼60% of VDJ/DJ-recombined pre-BII cells in which recruitment is not observed. However, this is not the case. Therefore, a more likely explanation is that, in allelically excluded cells, DJ-transcription alternates between ‘on' and ‘off' states where the ‘on' state is too infrequent to be detected by RNA FISH, although we know it is there because we detect the transcripts by RT–PCR.

Discussion

Our results demonstrate that during B-cell development, from the very earliest cells with both alleles in GL configuration, to those that have undergone IgH allelic exclusion, that is, from the pre-BII-cell stage and throughout subsequent B-cell development, up to and including activated splenic B cells, the predominant IgH transcription pattern is biallelic. In allelically excluded cells, this results in VDJ-transcription from both productively and nonproductively recombined VDJ-alleles as well as DJ-transcription from DJ-recombined alleles. We conclude that neither the establishment nor the maintenance of IgH allelic exclusion results in silencing of nonproductively recombined VDJ- or DJ-transcription.

Before VH to DJ-recombination, both IgH alleles are active and the observed biallelic transcription pattern is due to DJ-transcription, with Iμ-transcription also taking place in these cells. Similarly, biallelic transcription has also been observed in the Ig κ-locus before Vκ to Jκ-recombination (Singh et al, 2003). In cells that have undergone IgH allelic exclusion, DJ-transcription is detected by RT–PCR but not RNA FISH. However, this is not a consequence of the DJ-allele being recruited to centromeric heterochromatin as this association was only observed in less than half of VDJ/DJ-recombined cells. Biallelic transcription is not even observed in a monoclonal VDJ/DJ-recombined cell population, in which recruitment does not take place (Skok et al, 2001). It is therefore more likely that in allelically excluded cells, the DJ-allele experiences longer periods of transcriptional quiescence than before this event, and is consequently not detected by RNA FISH. The inability to detect primary transcripts by RNA FISH, but ability to detect RNA steady-state levels by RT–PCR has been previously reported, where germline VH sense transcripts are only detected by RT–PCR (Bolland et al, 2004). Nonetheless, that the DH-promoter is active throughout B-cell development is supported by its ability to drive the expression of a ‘reporter' gene (LMP2A) in more mature B cells, although its activity is lower than that of a VH promoter (Casola et al, 2004).

At the pre-BII stage, mono- and biallelic transcription is observed in an ∼1:1 ratio. A similar ratio is also observed in BM B and resting splenic B cells. That transcription takes place on both alleles in naïve splenic B cells is in agreement with earlier reports (Skok et al, 2001; Bolland et al, 2004), although our observation that activated splenic B cells also transcribe both IgH alleles disagrees with a previous report, which suggested a correlation between recruitment to centromeric heterochromatin and transcriptional silencing of both the DJ- and nonfunctional VDJ-allele (Skok et al, 2001). One possible explanation for this discrepancy may be the technique itself, that is, the use of different conditions for fixation, RNA versus single-stranded DNA probes. Nevertheless, biallelic IgH transcription is in agreement with the requirement for transcription in class switch recombination, which also takes place on both alleles (Radbruch et al, 1986). Moreover, somatic hypermutation, which represents a second wave of diversification of both IgH and IgL genes, also requires transcription, as shown by the importance of promoter and enhancer elements (Betz et al, 1994; Peters and Storb, 1996; Fukita et al, 1998). Furthermore, this process targets both the productive and nonproductive V(D)J-allele to a similar degree (Gonzalez-Fernandez et al, 1994), and requires a transcriptionally active promoter (Delpy et al, 2004).

Our data suggest that, upon establishment and maintenance of IgH allelic exclusion, the role of monoallelic recruitment to centromeric heterochromatin is not in silencing nonfunctional VDJ- or DJ-transcription. This is supported by our observation that, although one allele is associated with centromeric heterochromatin in about half of the pre-BII population, only half of the associated alleles are DJ. Consequently, the remaining must be either the functional or nonfunctional VDJ-allele, both of which are transcribed. However, it appears that the 5′-end of the IgH locus, that is, the VH region, is located closest to the centromeric domains, whereas the IgH constant region is 1–1.5 μm apart from the γ-satellite DNA in ∼30% of pre-BII cells (Roldan et al, 2005). This observation may also explain our slightly lower frequencies of associated alleles since our probes recognize the constant/intergenic region, which can be up to 2.5 Mb apart from the VH region, depending on recombination. It is thus possible that recruitment to heterochromatin, rather than repressing VDJ- and DJ-transcription, makes the VH region of the locus inaccessible to the recombination machinery and thereby prevents further recombination. Recruitment may still suppress transcription but rather VH germline-transcription as sense/antisense transcription over the VH region of nonrecombined VH gene segments is lost at the time of establishment of IgH allelic exclusion (Bolland et al, 2004).

It was recently reported that an IgH minilocus, containing premature translation-termination codons, that is, a nonfunctional VDJ, is transcriptionally silenced when expressed in HeLa cells (Buhler et al, 2005). However, as our data demonstrate that the nonfunctional VDJ-allele is transcriptionally active, this suggests that when expressed under control of its own regulatory elements it escapes this process. Furthermore, in normal B lymphocytes, it may not matter that the nonproductive VDJ-allele is transcribed as this allele does not produce a functional protein, hence the cell would still only express an antibody HC encoded by one of its two alleles and be allelically excluded.

Materials and methods

Mice

C57BL/6 (wt) (5 weeks), RAG1−/− (10 weeks) and μMT+/− (6 weeks) mice were used (Kitamura et al, 1991; Spanopoulou et al, 1994). All mice were kept under specific pathogen-free conditions and investigated under project licences PPL80/1501 and 1743 approved by the Home Office UK.

Cell lines

70Z/3 and 38C-13 (Nelson et al, 1983; Lennon and Perry, 1985, 1989) were cultured in RPMI with 5% fetal calf serum.

Primary cell preparations, sorting and in vitro cultures

BM and spleen cell suspensions were prepared using conventional techniques. BM cells were stained with anti-B220 in combination with mAbs specific for B-cell developmental stages; pre-BI, c-kit+; pre-BII, CD25+ or CD25+IgM−; B, IgM+, and sorted on a FACSDiVa™ or Aria™ (Becton Dickinson) as described previously (Parker et al, 2005). Purities within the nucleated cell gate were ∼95, >90 and ∼99% for pre-BI, pre-BII and B cells, respectively. RAG−/− BM cells were enriched using anti-CD19-beads (Milltenyi Biotech) according to the manufacturer's instructions with purities >95%. Splenic B cells were purified by CD43-depletion or CD19-enrichment (beads, Milltenyi Biotech) or sorting B220+IgM+. For in vitro stimulations, splenic B cells were enriched by CD43-depletion and cultured at 1 × 106 cells/ml in the presence of LPS (10 μg/ml, Sigma) and IL-4 (100 U/ml, Peprotech).

RNA preparation and RT–PCR

RNA preparation and cDNA synthesis have been described previously (Mundt et al, 2006). For primers and PCR conditions, see Supplementary Table 1.

Probes used for in situ hybridization

RNA FISH probes: VHJ558 (Bolland et al, 2004), CD45 (Skok et al, 2001), Iμ (∼1400 bp) similar to Bolland et al (2004) and μ (1150 bp) was established by PCR, using primers μs 5′-TCAAGGAACCTCAGTCACCG-3′, μas 5′-TCTCCAGTTTCG GCTGAATC-3′ followed by normal cloning procedures. Probe preparation has been described previously (Chakalova et al, 2004). DNA FISH probes: CR, ∼170 kb covering the Cδ-Cα region (BAC clone RPCl-24-258E20, Children's Hospital, Okland Research Institute, Okland, CA, USA); IR1, ∼60 kb (∼55 kb 5′ of to ∼5 kb 3′ of the most 5′ D) covering the VH to D intergenic region (mouse PAC Library RPC121, clone 449 G24, UK HGMP Resource Centre) and IR2, covering part of the VH to D intergenic region, consisting of a cocktail of seven plasmids with ∼1 kb inserts (Bolland D, manuscript in preparation). The IR2 probe was used in the (Brown, 2002) DNA FISH because the IR1 probe gave rise to high background levels. DNA FISH probes were nick translated in the presence of digoxygenin-dUTP (Roche) using standard procedures. The γ-satellite probe (Parker et al, 2005) was labeled with Alexa Fluor 594.

RNA, DNA and combined RNA/DNA FISH

RNA (Parker et al, 2005) and DNA (Ashe et al, 1997) FISH were carried out as described previously. For combined RNA/DNA FISH, the two protocols were combined (Ashe et al, 1997; Gribnau et al, 1998). In brief, cells were fixed as in the RNA FISH protocol. Both RNA and DNA FISH probes were denatured in hybridization mixture at 85°C for 5 min. DNA FISH probes were pre-annealed with mouse Cot-1 DNA (2 μg/slide) at 37°C. Slides were placed directly onto an 85°C heating block to denature the DNA. Probes were added to the slide and placed in a humidifying chamber, containing paper soaked in 50% formamide in 2 × SSC, and incubated for 16 h at 37°C. Probes were detected as described above. Images were analyzed under oil with a × 100 objective, using an Olympus BX40 fluorescence microscope. Typically, >100 nuclei were counted per slide in repeat experiments. DNA FISH preserving nuclear structure was performed as described previously (Brown, 2002). Images were analyzed by confocal microscopy on an Olympus FV1000 and analyzed using the FV10-ASW1.5 Viewer.

Supplementary Material

Supplementary Table 1

7601846s1.doc (36.5KB, doc)

Supplementary Figure 1

7601846s2.pdf (16.7KB, pdf)

Supplementary Figure 2

7601846s3.pdf (52.8KB, pdf)

Supplementary Figure 3

7601846s4.pdf (18.7KB, pdf)

Acknowledgments

We thank K Rajewsky (μMT) and A Corcoran (RAG1−/−) for mice, D Bolland and A Corcoran for VHJ558, Iμ and IR2 plasmids/probes, M Parker and L Chakalova for assistance with FISH and A Corcoran for critically reading the manuscript. This work was funded by the Biotechnology and Biological Sciences Research Council.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1

7601846s1.doc (36.5KB, doc)

Supplementary Figure 1

7601846s2.pdf (16.7KB, pdf)

Supplementary Figure 2

7601846s3.pdf (52.8KB, pdf)

Supplementary Figure 3

7601846s4.pdf (18.7KB, pdf)

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