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Silencing and nuclear repositioning of the lambda5 gene locus at the pre-B cell stage requires Aiolos and OBF-1.

Karnowski A, Cao C, Matthias G, Carotta S, Corcoran LM, Martensson IL, Skok JA, Matthias P - PLoS ONE (2008)

Bottom Line: However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes.Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos.These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland.

ABSTRACT
The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes lambda5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

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Silencing of λ5 expression at the pre-BII cell stage is impaired in absence of Aiolos and OBF-1.(A) λ5 expression in pre-BI and pre-BII cells of the different genotypes. Pre-BI cells (B220+ckit+IgM−), large or small pre-BII cells (B220+CD25+IgM−, discriminated on the basis of their FSC profile) were sorted from wild type, single- and double-deficient mice and λ5 expression was analyzed by real time RT-PCR. The histograms represent the mean±SE based on the analysis of at least three independent samples per genotype/stage. (B) Downregulation of λ5 expression in Aiolos−/−/OBF-1−/− splenic mature B cells. Small pre-BII (B220+CD25+IgM−) and splenic mature B (B220+IgMlowIgDhigh) cells of wild type, single- and double-deficient mice were sorted and the expression analysis was done by real time RT-PCR, as above. The histograms represent the mean±SE based on the analysis of two to three independent samples per genotype/stage. (C) Aiolos/OBF-1 double-deficient pre-BII cells fail to downregulate λ5 expression, but have a normal pre-BCR expression at the cell surface. Bone marrow cells from wild type, single- and double-mutant mice were stained for B220 together with surface pre-BCR (left panels) or intracellular surrogate light chain (λ5, right panels). Representative stainings are presented.
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pone-0003568-g004: Silencing of λ5 expression at the pre-BII cell stage is impaired in absence of Aiolos and OBF-1.(A) λ5 expression in pre-BI and pre-BII cells of the different genotypes. Pre-BI cells (B220+ckit+IgM−), large or small pre-BII cells (B220+CD25+IgM−, discriminated on the basis of their FSC profile) were sorted from wild type, single- and double-deficient mice and λ5 expression was analyzed by real time RT-PCR. The histograms represent the mean±SE based on the analysis of at least three independent samples per genotype/stage. (B) Downregulation of λ5 expression in Aiolos−/−/OBF-1−/− splenic mature B cells. Small pre-BII (B220+CD25+IgM−) and splenic mature B (B220+IgMlowIgDhigh) cells of wild type, single- and double-deficient mice were sorted and the expression analysis was done by real time RT-PCR, as above. The histograms represent the mean±SE based on the analysis of two to three independent samples per genotype/stage. (C) Aiolos/OBF-1 double-deficient pre-BII cells fail to downregulate λ5 expression, but have a normal pre-BCR expression at the cell surface. Bone marrow cells from wild type, single- and double-mutant mice were stained for B220 together with surface pre-BCR (left panels) or intracellular surrogate light chain (λ5, right panels). Representative stainings are presented.

Mentions: The combined expression data from pooled large and small pre-BII cells demonstrated strikingly elevated levels of VpreB1, 2 and λ5 mRNA in Aiolos single- and Aiolos/OBF-1 double-mutant mice. To determine whether this was the result of a net increase in transcription or a failure to properly silence these loci during development, λ5 mRNA expression was determined by quantitative RT-PCR in pre-BI cells, as well as in large or small pre-BII cells (Figure 4A). In wild type animals the λ5 gene was highly expressed at the pre-BI cell stage and subsequently downregulated more than ten-fold in large and small pre-BII cells, in agreement with previous results [6], [40]. Aiolos single-mutant mice showed normal λ5 mRNA levels in pre-BI cells, indicating that at this stage Aiolos does not influence expression of the λ5 gene. In contrast, in large or small pre-BII cells from Aiolos−/− mice, λ5 mRNA levels were reduced only two-fold compared to the pre-BI stage. The absence of OBF-1 led to a striking two-fold increase in λ5 expression in pre-BI cells, but the downregulation in large and small pre-BII cells was almost normal. Remarkably, the absence of both Aiolos and OBF-1 resulted in elevated λ5 mRNA levels in pre-BI cells, which were sustained in both large and small pre-BII cells (Figure 4A). In order to determine whether Aiolos−/−/OBF-1−/− mice are able to silence the expression of the λ5 gene at all, the expression of λ5 mRNA was determined in splenic mature resting B cells. As shown in Figure 4B, λ5 mRNA was not detected in splenic B cells, irrespective of genotype (See also Supplemental Figure S1A). Thus, an additional silencing mechanism exists, which is independent of Aiolos and OBF-1. In contrast to this, the Gelsolin gene, which also showed increased expression levels in the bone marrow of mutant mice, maintained elevated levels in splenic B cells (Supplemental Figure S1A–B). This indicates that in Aiolos−/−/OBF-1−/− mice, the few B cells that migrate to the spleen and develop into mature B cells, successfully silence the λ5 locus, but are still deregulated in the mRNA expression of other Aiolos/OBF-1 dependent genes.


Silencing and nuclear repositioning of the lambda5 gene locus at the pre-B cell stage requires Aiolos and OBF-1.

Karnowski A, Cao C, Matthias G, Carotta S, Corcoran LM, Martensson IL, Skok JA, Matthias P - PLoS ONE (2008)

Silencing of λ5 expression at the pre-BII cell stage is impaired in absence of Aiolos and OBF-1.(A) λ5 expression in pre-BI and pre-BII cells of the different genotypes. Pre-BI cells (B220+ckit+IgM−), large or small pre-BII cells (B220+CD25+IgM−, discriminated on the basis of their FSC profile) were sorted from wild type, single- and double-deficient mice and λ5 expression was analyzed by real time RT-PCR. The histograms represent the mean±SE based on the analysis of at least three independent samples per genotype/stage. (B) Downregulation of λ5 expression in Aiolos−/−/OBF-1−/− splenic mature B cells. Small pre-BII (B220+CD25+IgM−) and splenic mature B (B220+IgMlowIgDhigh) cells of wild type, single- and double-deficient mice were sorted and the expression analysis was done by real time RT-PCR, as above. The histograms represent the mean±SE based on the analysis of two to three independent samples per genotype/stage. (C) Aiolos/OBF-1 double-deficient pre-BII cells fail to downregulate λ5 expression, but have a normal pre-BCR expression at the cell surface. Bone marrow cells from wild type, single- and double-mutant mice were stained for B220 together with surface pre-BCR (left panels) or intracellular surrogate light chain (λ5, right panels). Representative stainings are presented.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571989&req=5

pone-0003568-g004: Silencing of λ5 expression at the pre-BII cell stage is impaired in absence of Aiolos and OBF-1.(A) λ5 expression in pre-BI and pre-BII cells of the different genotypes. Pre-BI cells (B220+ckit+IgM−), large or small pre-BII cells (B220+CD25+IgM−, discriminated on the basis of their FSC profile) were sorted from wild type, single- and double-deficient mice and λ5 expression was analyzed by real time RT-PCR. The histograms represent the mean±SE based on the analysis of at least three independent samples per genotype/stage. (B) Downregulation of λ5 expression in Aiolos−/−/OBF-1−/− splenic mature B cells. Small pre-BII (B220+CD25+IgM−) and splenic mature B (B220+IgMlowIgDhigh) cells of wild type, single- and double-deficient mice were sorted and the expression analysis was done by real time RT-PCR, as above. The histograms represent the mean±SE based on the analysis of two to three independent samples per genotype/stage. (C) Aiolos/OBF-1 double-deficient pre-BII cells fail to downregulate λ5 expression, but have a normal pre-BCR expression at the cell surface. Bone marrow cells from wild type, single- and double-mutant mice were stained for B220 together with surface pre-BCR (left panels) or intracellular surrogate light chain (λ5, right panels). Representative stainings are presented.
Mentions: The combined expression data from pooled large and small pre-BII cells demonstrated strikingly elevated levels of VpreB1, 2 and λ5 mRNA in Aiolos single- and Aiolos/OBF-1 double-mutant mice. To determine whether this was the result of a net increase in transcription or a failure to properly silence these loci during development, λ5 mRNA expression was determined by quantitative RT-PCR in pre-BI cells, as well as in large or small pre-BII cells (Figure 4A). In wild type animals the λ5 gene was highly expressed at the pre-BI cell stage and subsequently downregulated more than ten-fold in large and small pre-BII cells, in agreement with previous results [6], [40]. Aiolos single-mutant mice showed normal λ5 mRNA levels in pre-BI cells, indicating that at this stage Aiolos does not influence expression of the λ5 gene. In contrast, in large or small pre-BII cells from Aiolos−/− mice, λ5 mRNA levels were reduced only two-fold compared to the pre-BI stage. The absence of OBF-1 led to a striking two-fold increase in λ5 expression in pre-BI cells, but the downregulation in large and small pre-BII cells was almost normal. Remarkably, the absence of both Aiolos and OBF-1 resulted in elevated λ5 mRNA levels in pre-BI cells, which were sustained in both large and small pre-BII cells (Figure 4A). In order to determine whether Aiolos−/−/OBF-1−/− mice are able to silence the expression of the λ5 gene at all, the expression of λ5 mRNA was determined in splenic mature resting B cells. As shown in Figure 4B, λ5 mRNA was not detected in splenic B cells, irrespective of genotype (See also Supplemental Figure S1A). Thus, an additional silencing mechanism exists, which is independent of Aiolos and OBF-1. In contrast to this, the Gelsolin gene, which also showed increased expression levels in the bone marrow of mutant mice, maintained elevated levels in splenic B cells (Supplemental Figure S1A–B). This indicates that in Aiolos−/−/OBF-1−/− mice, the few B cells that migrate to the spleen and develop into mature B cells, successfully silence the λ5 locus, but are still deregulated in the mRNA expression of other Aiolos/OBF-1 dependent genes.

Bottom Line: However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes.Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos.These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland.

ABSTRACT
The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes lambda5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

Show MeSH
Related in: MedlinePlus