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. 2005 Nov 16;24(22):3895-905.
doi: 10.1038/sj.emboj.7600850. Epub 2005 Nov 10.

The pre-B-cell Receptor Induces Silencing of VpreB and lambda5 Transcription

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Free PMC article

The pre-B-cell Receptor Induces Silencing of VpreB and lambda5 Transcription

Mathew J Parker et al. EMBO J. .
Free PMC article

Abstract

The pre-B-cell receptor (pre-BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre-BII-cell proliferative expansion. We have investigated whether the pre-BCR also signals downregulation of the SLC genes (VpreB and lambda5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre-BI to large pre-BII-cell stage, there is a shift from bi- to mono-allelic lambda5 transcription, while the second allele is silenced in small pre-BII cells. A VpreB1-promoter-driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre-BCR-deficient mice show a temporal delay in lambda5 downregulation, thereby demonstrating that the pre-BCR is essential for monoallelic silencing at the large pre-BII-cell stage. Our data also suggest that SLP-65 is one of the signaling components important for this process. Furthermore, the VpreB1/lambda5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.

Figures

Figure 1
Figure 1
Pro- and pre-BCR expression in ex vivo mouse BM B cells. (A) B-cell development showing expression of the pro-BCR (SLC with glycoproteins), pre-BCR (SLC with μHC) and BCR (μHC with IgL chain). Large cells are cycling and small cells are noncycling. Cell surface markers used are also shown. All cells are B220+CD19+. (B) CD19-enriched BM cells were gated (G1) on pre-BI cells and analyzed for surface expression of SLC, pre-BCR or isotype control, (e) indicates that the EAS kit was used. As the levels of λ5 (shown) and VpreB were similar, λ5+ cells represent SLC+ cells. (C) BM cells were gated (G1) on pro-/pre-BI cells and analyzed for intracellular expression of SLC and μHC. (D) As in (B), except that cells were gated (G1) on pre-BII cells. (E) As in (C), except that cells were gated (G1) on pre-BII cells and size. Representative results from at least three experiments.
Figure 2
Figure 2
Transcription patterns of the λ5 and CD45 genes during B-cell development. (A) Schematic diagram of the VpreB1-λ5 locus. The position of the λ5 probe is indicated. (B) RNA FISH. Percentages (±s.e.m.) of nuclei with CD45 or λ5 signals in sorted BM cells from indicated stages. (C) Percentages (±s.d.) of signal-positive nuclei containing either one, two or four signals at the pre-BI and large pre-BII stages. A total of >900 nuclei were counted for each stage (>3 experiments). (D) Typical images of nuclei hybridized with either the CD45 or λ5 probe (green), in combination with an antibody recognizing PCNA (red), distinguishing S-phase nuclei. DNA was counterstained with DAPI (blue) to verify nuclear integrity. For CD45, typically two (G1 phase) or four (S phase) foci were observed in both pre-BI and pre-BII cells. While two (G1) or four (S) λ5 foci were also observed in pre-BI cells, only one (G1) or two (S) were observed in large pre-BII cells.
Figure 3
Figure 3
Transcription pattern of the λ5 gene after induction of pre-BCR expression. (A) FACS analysis of CD19-enriched BM pre-BI cells from tet-μH mice before and at the indicated times following the in vitro induction of μHC expression. The following is shown: surface expression of SLC and μHC using EAS (e) and cytoplasmic μH expression; cell size at days 0 and 4 (dotted and filled lines, respectively, in histogram). (B) RNA FISH on the cells in (A) using either CD45 or λ5 probes daily following induction of TG μHC, that is, pre-BCR, expression. Percentages of nuclei with signals are shown. (C) Percentages of PCNA+ signal-positive nuclei containing one, two or four signals at the indicated times. The data from one representative experiment (out of three) are shown. A total of >900 nuclei were counted for each time point.
Figure 4
Figure 4
Transcription pattern of the −214VpreB1-HuCD122 transgene during B-cell development. RNA FISH analysis of ex vivo BM pre-BI and large pre-BII cells from −214VpreB1-HuCD122+/+ TG mice, in which the reporter gene is driven by the VpreB1 promoter. The percentages of signal-positive nuclei with one, two or four TG or λ5 signals are shown. For the TG, a total of >200 nuclei was counted for each stage (two experiments).
Figure 5
Figure 5
RNA FISH analysis of transitional pre-BI cells from normal mice. (A) Sorted ex vivo BM transitional pre-BI cells. (B) In vitro cultured transitional pre-BI cells. Total pre-BI cells before culture were included for comparison. The percentages of signal-positive nuclei with one, two or four signals are shown using the indicated probes. The results from (A) two experiments (±s.d.) and (B) one experiment are shown. A total of >300 nuclei were counted for each population.
Figure 6
Figure 6
Analysis of pre-BCR-deficient pre-BI and pre-BII cells. (A) CD19-enriched BM cells from VpreB−/− mice were sorted as pre-BI and pre-BII cells. (B) RNA FISH analysis of the cells in (A). The percentages (±s.d.) of signal-positive nuclei with one, two or four signals using the indicated probes are shown. A total of >600 nuclei were counted for each population (at least two experiments).
Figure 7
Figure 7
RNA FISH analysis of pre-B cells lacking SLP-65. (A) The percentages (±s.e.m.) of signal-positive nuclei with one, two or four signals using the indicated probes are shown for the SLP-65−/− cell line, 26.5. (B) Percentages (±s.d.) of nuclei with CD45 or λ5 signals are shown for pre-BCR+ and small pre-BII cells from SLP-65−/− mice. (C) Percentages (±s.d.) of signal-positive nuclei containing one, two or four signals in pre-BCR+ cells. (A–C) A total of >300 nuclei were counted (at least two experiments).
Figure 8
Figure 8
DNA FISH analysis of BM and splenic B cells from wt and pre-BCR-deficient mice. (A) Confocal section images of nuclei after DNA FISH are shown, combining the λ5 locus probe (red) with a probe detecting γ-satellite DNA (green). Images demonstrate association between a single, both or neither λ5 allele and γ-satellite DNA. Alleles 1 and 2 indicate the image plane where the respective allele is observed. (B) Percentages of nuclei with a single, both or neither λ5 allele associated with γ-satellite DNA in the indicated cell populations from wt mice. Association was scored when an allele was in apparent contact with γ-satellite foci. (C) As in (B), except that cells were from VpreB−/− mice. (D) Percentages of alleles located at the nuclear periphery in the indicated cell populations. Cell populations were from wt (left) and VpreB−/− (right) mice. (E) Immuno-DNA FISH analysis of wt splenic B cells using the λ5 locus probe (green) and staining with antibodies specific to the amino- and carboxy-terminal portions of lamin B (red).

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