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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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The transition from small pre-BII to immature B cells is impaired in Aio−/−/OBF-1−/− mice.Flow cytometry analysis of bone marrow cells from 6–10 weeks old control wild type, single- and double-deficient mice. (A) Analysis of B220+ IgM+ IgD− immature B cells in the bone marrow. Cells were stained with anti-B220-APC, anti-IgM-FITC, anti-IgD-Biotin followed by Streptavidin-PE. Only IgD− cells are displayed and the percentages indicated are relative to the IgD− cells. The percentage of IgD− cells from total cells are as follows: WT, 89%; Aiolos−/−, 88%; OBF-1−/−, 92%; Aio−/−/OBF-1−/−, 95%. (B) In the left part detection of B220+ CD25+ pre-BII B cells. In the right part, pre-BII (B220+CD25+) cells were gated and their FSC analyzed. Percentages indicate the proportion of small or large cells. Data are presented from one representative experiment, out of three. (C) Pre-BII cells (B220+ CD25+ IgM−) were FACS sorted from wild type and Aiolos/OBF-1 double-deficient mice by flow cytometry. Serial dilutions of the genomic DNA from the sorted cells were analyzed by PCR using primers detecting the indicated κ and λ light chain rearrangements. The relative amount of DNA used in the reaction was adjusted according to a real time PCR for the 18S gene (not shown).
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pone-0003568-g001: The transition from small pre-BII to immature B cells is impaired in Aio−/−/OBF-1−/− mice.Flow cytometry analysis of bone marrow cells from 6–10 weeks old control wild type, single- and double-deficient mice. (A) Analysis of B220+ IgM+ IgD− immature B cells in the bone marrow. Cells were stained with anti-B220-APC, anti-IgM-FITC, anti-IgD-Biotin followed by Streptavidin-PE. Only IgD− cells are displayed and the percentages indicated are relative to the IgD− cells. The percentage of IgD− cells from total cells are as follows: WT, 89%; Aiolos−/−, 88%; OBF-1−/−, 92%; Aio−/−/OBF-1−/−, 95%. (B) In the left part detection of B220+ CD25+ pre-BII B cells. In the right part, pre-BII (B220+CD25+) cells were gated and their FSC analyzed. Percentages indicate the proportion of small or large cells. Data are presented from one representative experiment, out of three. (C) Pre-BII cells (B220+ CD25+ IgM−) were FACS sorted from wild type and Aiolos/OBF-1 double-deficient mice by flow cytometry. Serial dilutions of the genomic DNA from the sorted cells were analyzed by PCR using primers detecting the indicated κ and λ light chain rearrangements. The relative amount of DNA used in the reaction was adjusted according to a real time PCR for the 18S gene (not shown).

Mentions: The loss of either Aiolos or OBF-1 results in a reduction of immature splenic B cells and impaired formation of MZ B cells [10], [15], [22]. We have shown previously that the number of mature resting B cells is only marginally altered in the single-mutant mice, but is reduced several-fold in Aiolos−/−/OBF-1−/− mice [21]. To better understand the molecular basis of this phenomenon, we have examined here early B cell development in mice of the different genotypes. In agreement with our previous studies [21], we found that immature B cells (B220+ IgM+ IgD−) are severely reduced in Aiolos/OBF-1 double - but not single-deficient or wild type mice (Figure 1A). As previously reported [27], Aiolos single-deficient mice showed an increase in the proportion of pre-BII (B220+ CD25+ IgM−) cells. Moreover, the double-deficient mice exhibited a further small increase in the proportion of pre-BII cells, although OBF-1 single-deficient mice showed no change in this cell population (Figure 1B). To distinguish between the dividing and non-dividing, but light chain rearranging cells, pre-BII cells can be fractionated on the basis of their size (FSC). Independent of genotype, about 80 to 90% of the pre-BII cell population represented small non-dividing cells (Figure 1B), which was also confirmed by DNA content analysis from sorted pre-BII cell populations (data not shown).


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)

The transition from small pre-BII to immature B cells is impaired in Aio−/−/OBF-1−/− mice.Flow cytometry analysis of bone marrow cells from 6–10 weeks old control wild type, single- and double-deficient mice. (A) Analysis of B220+ IgM+ IgD− immature B cells in the bone marrow. Cells were stained with anti-B220-APC, anti-IgM-FITC, anti-IgD-Biotin followed by Streptavidin-PE. Only IgD− cells are displayed and the percentages indicated are relative to the IgD− cells. The percentage of IgD− cells from total cells are as follows: WT, 89%; Aiolos−/−, 88%; OBF-1−/−, 92%; Aio−/−/OBF-1−/−, 95%. (B) In the left part detection of B220+ CD25+ pre-BII B cells. In the right part, pre-BII (B220+CD25+) cells were gated and their FSC analyzed. Percentages indicate the proportion of small or large cells. Data are presented from one representative experiment, out of three. (C) Pre-BII cells (B220+ CD25+ IgM−) were FACS sorted from wild type and Aiolos/OBF-1 double-deficient mice by flow cytometry. Serial dilutions of the genomic DNA from the sorted cells were analyzed by PCR using primers detecting the indicated κ and λ light chain rearrangements. The relative amount of DNA used in the reaction was adjusted according to a real time PCR for the 18S gene (not shown).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571989&req=5

pone-0003568-g001: The transition from small pre-BII to immature B cells is impaired in Aio−/−/OBF-1−/− mice.Flow cytometry analysis of bone marrow cells from 6–10 weeks old control wild type, single- and double-deficient mice. (A) Analysis of B220+ IgM+ IgD− immature B cells in the bone marrow. Cells were stained with anti-B220-APC, anti-IgM-FITC, anti-IgD-Biotin followed by Streptavidin-PE. Only IgD− cells are displayed and the percentages indicated are relative to the IgD− cells. The percentage of IgD− cells from total cells are as follows: WT, 89%; Aiolos−/−, 88%; OBF-1−/−, 92%; Aio−/−/OBF-1−/−, 95%. (B) In the left part detection of B220+ CD25+ pre-BII B cells. In the right part, pre-BII (B220+CD25+) cells were gated and their FSC analyzed. Percentages indicate the proportion of small or large cells. Data are presented from one representative experiment, out of three. (C) Pre-BII cells (B220+ CD25+ IgM−) were FACS sorted from wild type and Aiolos/OBF-1 double-deficient mice by flow cytometry. Serial dilutions of the genomic DNA from the sorted cells were analyzed by PCR using primers detecting the indicated κ and λ light chain rearrangements. The relative amount of DNA used in the reaction was adjusted according to a real time PCR for the 18S gene (not shown).
Mentions: The loss of either Aiolos or OBF-1 results in a reduction of immature splenic B cells and impaired formation of MZ B cells [10], [15], [22]. We have shown previously that the number of mature resting B cells is only marginally altered in the single-mutant mice, but is reduced several-fold in Aiolos−/−/OBF-1−/− mice [21]. To better understand the molecular basis of this phenomenon, we have examined here early B cell development in mice of the different genotypes. In agreement with our previous studies [21], we found that immature B cells (B220+ IgM+ IgD−) are severely reduced in Aiolos/OBF-1 double - but not single-deficient or wild type mice (Figure 1A). As previously reported [27], Aiolos single-deficient mice showed an increase in the proportion of pre-BII (B220+ CD25+ IgM−) cells. Moreover, the double-deficient mice exhibited a further small increase in the proportion of pre-BII cells, although OBF-1 single-deficient mice showed no change in this cell population (Figure 1B). To distinguish between the dividing and non-dividing, but light chain rearranging cells, pre-BII cells can be fractionated on the basis of their size (FSC). Independent of genotype, about 80 to 90% of the pre-BII cell population represented small non-dividing cells (Figure 1B), which was also confirmed by DNA content analysis from sorted pre-BII cell populations (data not shown).

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