Limits...
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.

Show MeSH
Identification of genes regulated by Aiolos and OBF-1 in pre-BII cells.Gene expression profiles in B220+CD25+ pre-BII cells were determined by MOE430a Affymetrix GeneChip; for each genotype two RNA samples were prepared from independent pools of mice and microarray analysis was done in duplicate. (A) 48 genes showed a 3 fold expression changes in single- or double-mutant pre-BII cells compared to pre-BII cells from wild type mice (p-value cutoff: 0.05). The genes are grouped according to their expression profile in pre-BII cells from all genotypes. Low mRNA expression, blue; high mRNA expression, red. (B) Expression of genes that have been reported previously to be dependent on OBF-1 expression: Myla, Ms4a1, S100a10. (C) Expression of genes that show a strong expression increase specifically in Aio−/−/OBF-1−/− pre-B cells: Ramp1, Gpr49, Gelsolin (Gsn). (D) Expression of the surrogate light chain genes: λ5, VpreB1 and VpreB2. Figures show raw Affymetrix expression score after array normalization.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2571989&req=5

pone-0003568-g003: Identification of genes regulated by Aiolos and OBF-1 in pre-BII cells.Gene expression profiles in B220+CD25+ pre-BII cells were determined by MOE430a Affymetrix GeneChip; for each genotype two RNA samples were prepared from independent pools of mice and microarray analysis was done in duplicate. (A) 48 genes showed a 3 fold expression changes in single- or double-mutant pre-BII cells compared to pre-BII cells from wild type mice (p-value cutoff: 0.05). The genes are grouped according to their expression profile in pre-BII cells from all genotypes. Low mRNA expression, blue; high mRNA expression, red. (B) Expression of genes that have been reported previously to be dependent on OBF-1 expression: Myla, Ms4a1, S100a10. (C) Expression of genes that show a strong expression increase specifically in Aio−/−/OBF-1−/− pre-B cells: Ramp1, Gpr49, Gelsolin (Gsn). (D) Expression of the surrogate light chain genes: λ5, VpreB1 and VpreB2. Figures show raw Affymetrix expression score after array normalization.

Mentions: We next investigated by quantitative RT-PCR the expression pattern of OBF-1 and Aiolos during B cell development. Both genes showed a similar pattern of expression: low in pre-BI, intermediate in pre-BII, high in immature B and intermediate in mature B cells (Figure 2), thus supporting a role for these factors in early B cell development. In order to determine the role of OBF-1 and Aiolos in pre-BII cells, the mRNA expression profile in pre-BII cells from wild type, Aiolos or OBF-1 single-mutant and double-mutant mice were measured, using the MOE430A Affymetrix GeneChip (Figure 3A). Previous mRNA expression analysis of OBF-1 deficient B cells have indicated a role for this factor in regulating several genes, including Lck, Kcnn4, cdc37, Myla, Ms4a11 or S100a10 [36], [37], some of which were confirmed by our present analysis (see Figure 3B). In comparing the transcriptomes in cells of the four genotypes we found that the majority of genes with a reduced mRNA expression in the double-mutant pre-BII showed a similarly diminished expression in OBF-1−/− pre-BII cells. Most of the genes that were upregulated in the double-mutant pre-BII, were also partially upregulated in pre-BII cells from either single-mutant mice. We found that the expression of some genes, like RAMP1 (calcitonin receptor-activity modifying protein 1), Gpr49 (a G-protein-coupled receptor), or Gsn (Gelsolin, an actin binding and severing protein) was found to be synergistically upregulated in the pre-BII cells from double-deficient mice (Figure 3C). The mRNA expression of the major players in immunoglobulin gene rearrangement (RAG1-2, dntt, Ku70, polm) was not altered by the absence of Aiolos, OBF-1 or both factors (data not shown). Strikingly, loss of Aiolos and OBF-1 altered the expression of the genes encoding the SLC, λ5, VpreB1 and VpreB2 (Figure 3D). The role of Ikaros and Aiolos in regulating expression of λ5 was recently demonstrated [38], [39]. This regulatory role was confirmed by our observations of a 10-fold increase of λ5 mRNA expression levels in Aiolos deficient pre-BII cells. Furthermore, the mRNA expression levels of VpreB1 and VpreB2 were increased up to 7-fold in Aiolos deficient cells, thus demonstrating a regulatory role of Aiolos for all members of the surrogate light chain. Although λ5 and VpreB1,2 mRNA levels were only moderately elevated (2-fold) in OBF-1 deficient pre-BII cells, expression of these genes was increased up to 25-fold in pre-BII cells of the double-mutant mice (Figure 3D) indicating that λ5, VpreB1 and VpreB2 depend on both Aiolos and OBF-1 for their correct expression.


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)

Identification of genes regulated by Aiolos and OBF-1 in pre-BII cells.Gene expression profiles in B220+CD25+ pre-BII cells were determined by MOE430a Affymetrix GeneChip; for each genotype two RNA samples were prepared from independent pools of mice and microarray analysis was done in duplicate. (A) 48 genes showed a 3 fold expression changes in single- or double-mutant pre-BII cells compared to pre-BII cells from wild type mice (p-value cutoff: 0.05). The genes are grouped according to their expression profile in pre-BII cells from all genotypes. Low mRNA expression, blue; high mRNA expression, red. (B) Expression of genes that have been reported previously to be dependent on OBF-1 expression: Myla, Ms4a1, S100a10. (C) Expression of genes that show a strong expression increase specifically in Aio−/−/OBF-1−/− pre-B cells: Ramp1, Gpr49, Gelsolin (Gsn). (D) Expression of the surrogate light chain genes: λ5, VpreB1 and VpreB2. Figures show raw Affymetrix expression score after array normalization.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003568-g003: Identification of genes regulated by Aiolos and OBF-1 in pre-BII cells.Gene expression profiles in B220+CD25+ pre-BII cells were determined by MOE430a Affymetrix GeneChip; for each genotype two RNA samples were prepared from independent pools of mice and microarray analysis was done in duplicate. (A) 48 genes showed a 3 fold expression changes in single- or double-mutant pre-BII cells compared to pre-BII cells from wild type mice (p-value cutoff: 0.05). The genes are grouped according to their expression profile in pre-BII cells from all genotypes. Low mRNA expression, blue; high mRNA expression, red. (B) Expression of genes that have been reported previously to be dependent on OBF-1 expression: Myla, Ms4a1, S100a10. (C) Expression of genes that show a strong expression increase specifically in Aio−/−/OBF-1−/− pre-B cells: Ramp1, Gpr49, Gelsolin (Gsn). (D) Expression of the surrogate light chain genes: λ5, VpreB1 and VpreB2. Figures show raw Affymetrix expression score after array normalization.
Mentions: We next investigated by quantitative RT-PCR the expression pattern of OBF-1 and Aiolos during B cell development. Both genes showed a similar pattern of expression: low in pre-BI, intermediate in pre-BII, high in immature B and intermediate in mature B cells (Figure 2), thus supporting a role for these factors in early B cell development. In order to determine the role of OBF-1 and Aiolos in pre-BII cells, the mRNA expression profile in pre-BII cells from wild type, Aiolos or OBF-1 single-mutant and double-mutant mice were measured, using the MOE430A Affymetrix GeneChip (Figure 3A). Previous mRNA expression analysis of OBF-1 deficient B cells have indicated a role for this factor in regulating several genes, including Lck, Kcnn4, cdc37, Myla, Ms4a11 or S100a10 [36], [37], some of which were confirmed by our present analysis (see Figure 3B). In comparing the transcriptomes in cells of the four genotypes we found that the majority of genes with a reduced mRNA expression in the double-mutant pre-BII showed a similarly diminished expression in OBF-1−/− pre-BII cells. Most of the genes that were upregulated in the double-mutant pre-BII, were also partially upregulated in pre-BII cells from either single-mutant mice. We found that the expression of some genes, like RAMP1 (calcitonin receptor-activity modifying protein 1), Gpr49 (a G-protein-coupled receptor), or Gsn (Gelsolin, an actin binding and severing protein) was found to be synergistically upregulated in the pre-BII cells from double-deficient mice (Figure 3C). The mRNA expression of the major players in immunoglobulin gene rearrangement (RAG1-2, dntt, Ku70, polm) was not altered by the absence of Aiolos, OBF-1 or both factors (data not shown). Strikingly, loss of Aiolos and OBF-1 altered the expression of the genes encoding the SLC, λ5, VpreB1 and VpreB2 (Figure 3D). The role of Ikaros and Aiolos in regulating expression of λ5 was recently demonstrated [38], [39]. This regulatory role was confirmed by our observations of a 10-fold increase of λ5 mRNA expression levels in Aiolos deficient pre-BII cells. Furthermore, the mRNA expression levels of VpreB1 and VpreB2 were increased up to 7-fold in Aiolos deficient cells, thus demonstrating a regulatory role of Aiolos for all members of the surrogate light chain. Although λ5 and VpreB1,2 mRNA levels were only moderately elevated (2-fold) in OBF-1 deficient pre-BII cells, expression of these genes was increased up to 25-fold in pre-BII cells of the double-mutant mice (Figure 3D) indicating that λ5, VpreB1 and VpreB2 depend on both Aiolos and OBF-1 for their correct expression.

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