Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Oct 3;26(19):4273-82.
doi: 10.1038/sj.emboj.7601846. Epub 2007 Sep 6.

Transcription of Productive and Nonproductive VDJ-recombined Alleles After IgH Allelic Exclusion

Affiliations
Free PMC article

Transcription of Productive and Nonproductive VDJ-recombined Alleles After IgH Allelic Exclusion

Janssen Daly et al. EMBO J. .
Free PMC article

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.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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).
Figure 3
Figure 3
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).
Figure 4
Figure 4
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.
Figure 5
Figure 5
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+).
Figure 6
Figure 6
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.
Figure 7
Figure 7
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.

Similar articles

See all similar articles

Cited by 15 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback