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Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components.

Galler GR, Mundt C, Parker M, Pelanda R, Mårtensson IL, Winkler TH - J. Exp. Med. (2004)

Bottom Line: Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion.It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele.Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells.

View Article: PubMed Central - PubMed

Affiliation: Hematopoiesis Unit, Nikolaus-Fiebiger-Center, Friedrich-Alexander University, Glueckstrasse 6, 91054 Erlangen, Germany.

ABSTRACT
Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

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Induction of μHC in pro–B cells induces down-regulation of Rag1 primary transcripts in the absence of λ5. CD19+ BM cells from tet-μHC and tet-μHC λ5−/− mice were isolated by MACS and cultured on stromal cells with or without Tet for 12 h. Cells were fixed onto slides and subjected to RNA FISH to detect primary transcripts on individual alleles in single nuclei using ssDNA probes specific for Rag1 and CD45 (control). Hybridized probes were detected and visualized by fluorochrome-labeled Abs, and cells displaying signals were counted (top, >150 nuclei per slide). Induction of cytoplasmic μHC expression was verified by intracellular FACS® (middle, unshaded). Nuclei were counterstained with 4,6-diamino-2-phenylindole. Microscopical images (bottom) show representative nuclei hybridized for Rag1 or CD45, respectively. The experiment was repeated with comparable results.
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fig4: Induction of μHC in pro–B cells induces down-regulation of Rag1 primary transcripts in the absence of λ5. CD19+ BM cells from tet-μHC and tet-μHC λ5−/− mice were isolated by MACS and cultured on stromal cells with or without Tet for 12 h. Cells were fixed onto slides and subjected to RNA FISH to detect primary transcripts on individual alleles in single nuclei using ssDNA probes specific for Rag1 and CD45 (control). Hybridized probes were detected and visualized by fluorochrome-labeled Abs, and cells displaying signals were counted (top, >150 nuclei per slide). Induction of cytoplasmic μHC expression was verified by intracellular FACS® (middle, unshaded). Nuclei were counterstained with 4,6-diamino-2-phenylindole. Microscopical images (bottom) show representative nuclei hybridized for Rag1 or CD45, respectively. The experiment was repeated with comparable results.

Mentions: Transgenic μHC expression is already detectable 12 h after withdrawal of Tet (4), but Rag-driven GFP down-regulation was not observed until 48 h. Therefore, to determine directly whether μHC mediates termination of Rag transcription and whether this has already taken place after 12 h, RNA FISH was performed (15). The advantage is that this technique allows the detection of RNA produced at the site of transcription (i.e., at individual alleles, in single cells [nuclei]). For this, we made a probe that specifically detects Rag1 primary transcripts (Fig. 4 and not depicted). As a control, we used a probe that detects CD45, which is expressed on both pro–B and pre–B cells and, hence, is independent of μHC. The CD45 gene is biallelically transcribed (27). Numbers of nuclei showing 0, 1, or 2 foci of transcription were counted (>150 nuclei per slide). Nuclei with one CD45 probe signal may reflect an oscillating transcriptional status of this gene (28, 29). We assume that this is also the case for the Rag gene because a comparable number of nuclei (5–10%) were observed with only one detectable CD45 or Rag1 signal. Therefore, percentages of nuclei with one or two signals were combined.


Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components.

Galler GR, Mundt C, Parker M, Pelanda R, Mårtensson IL, Winkler TH - J. Exp. Med. (2004)

Induction of μHC in pro–B cells induces down-regulation of Rag1 primary transcripts in the absence of λ5. CD19+ BM cells from tet-μHC and tet-μHC λ5−/− mice were isolated by MACS and cultured on stromal cells with or without Tet for 12 h. Cells were fixed onto slides and subjected to RNA FISH to detect primary transcripts on individual alleles in single nuclei using ssDNA probes specific for Rag1 and CD45 (control). Hybridized probes were detected and visualized by fluorochrome-labeled Abs, and cells displaying signals were counted (top, >150 nuclei per slide). Induction of cytoplasmic μHC expression was verified by intracellular FACS® (middle, unshaded). Nuclei were counterstained with 4,6-diamino-2-phenylindole. Microscopical images (bottom) show representative nuclei hybridized for Rag1 or CD45, respectively. The experiment was repeated with comparable results.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211789&req=5

fig4: Induction of μHC in pro–B cells induces down-regulation of Rag1 primary transcripts in the absence of λ5. CD19+ BM cells from tet-μHC and tet-μHC λ5−/− mice were isolated by MACS and cultured on stromal cells with or without Tet for 12 h. Cells were fixed onto slides and subjected to RNA FISH to detect primary transcripts on individual alleles in single nuclei using ssDNA probes specific for Rag1 and CD45 (control). Hybridized probes were detected and visualized by fluorochrome-labeled Abs, and cells displaying signals were counted (top, >150 nuclei per slide). Induction of cytoplasmic μHC expression was verified by intracellular FACS® (middle, unshaded). Nuclei were counterstained with 4,6-diamino-2-phenylindole. Microscopical images (bottom) show representative nuclei hybridized for Rag1 or CD45, respectively. The experiment was repeated with comparable results.
Mentions: Transgenic μHC expression is already detectable 12 h after withdrawal of Tet (4), but Rag-driven GFP down-regulation was not observed until 48 h. Therefore, to determine directly whether μHC mediates termination of Rag transcription and whether this has already taken place after 12 h, RNA FISH was performed (15). The advantage is that this technique allows the detection of RNA produced at the site of transcription (i.e., at individual alleles, in single cells [nuclei]). For this, we made a probe that specifically detects Rag1 primary transcripts (Fig. 4 and not depicted). As a control, we used a probe that detects CD45, which is expressed on both pro–B and pre–B cells and, hence, is independent of μHC. The CD45 gene is biallelically transcribed (27). Numbers of nuclei showing 0, 1, or 2 foci of transcription were counted (>150 nuclei per slide). Nuclei with one CD45 probe signal may reflect an oscillating transcriptional status of this gene (28, 29). We assume that this is also the case for the Rag gene because a comparable number of nuclei (5–10%) were observed with only one detectable CD45 or Rag1 signal. Therefore, percentages of nuclei with one or two signals were combined.

Bottom Line: Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion.It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele.Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells.

View Article: PubMed Central - PubMed

Affiliation: Hematopoiesis Unit, Nikolaus-Fiebiger-Center, Friedrich-Alexander University, Glueckstrasse 6, 91054 Erlangen, Germany.

ABSTRACT
Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

Show MeSH
Related in: MedlinePlus