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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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Assessing the role of VpreB1 and λ5 expression for pro B cells maturation in vitro.(A) In the upper part a schematic of the retroviral construct used to transduce pro/pre-B cells is presented. The control construct is identical, but only contains EGFP. (B) WEHI231 lymphoma B cells were transduced with either VpreB1-IRES-λ5-EGFP or an EGFP retroviral expression construct. Expression of VpreB1 and λ5 was determined by staining for surface expression of the pre-BCR (SL156) and λ5 (LM34). Possible displacement of κLC on transduced WEHI231 cells was determined by staining for surface κLC. Representative stainings are presented. (C) Schematic of the transduction experiment with bone marrow cells. Hematopoietic stem cells (HSC) were first transduced with a Bcl-2 expressing retrovirus to enhance their survival and were differentiated into pro/preB cultures in the presence of Flt3 and IL-7. Subsequently, these cultures were transduced with the indicated retroviruses and differentiated in vitro by withdrawing IL-7.
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pone-0003568-g007: Assessing the role of VpreB1 and λ5 expression for pro B cells maturation in vitro.(A) In the upper part a schematic of the retroviral construct used to transduce pro/pre-B cells is presented. The control construct is identical, but only contains EGFP. (B) WEHI231 lymphoma B cells were transduced with either VpreB1-IRES-λ5-EGFP or an EGFP retroviral expression construct. Expression of VpreB1 and λ5 was determined by staining for surface expression of the pre-BCR (SL156) and λ5 (LM34). Possible displacement of κLC on transduced WEHI231 cells was determined by staining for surface κLC. Representative stainings are presented. (C) Schematic of the transduction experiment with bone marrow cells. Hematopoietic stem cells (HSC) were first transduced with a Bcl-2 expressing retrovirus to enhance their survival and were differentiated into pro/preB cultures in the presence of Flt3 and IL-7. Subsequently, these cultures were transduced with the indicated retroviruses and differentiated in vitro by withdrawing IL-7.

Mentions: The expression of members of the surrogate light chain VpreB1, 2 and λ5 is tightly controlled at the transition from pre-BI, large and small pre-BII, to immature B cells, suggesting that this downregulation is essential. In order to test whether high levels of the surrogate light chain could interfere with the maturation of early B cell stages, we set out to overexpress λ5 and VpreB1 in early B cells, using a λ5-IRES-VpreB1-EGFP retroviral expression vector (Figure 7A). First, expression of VpreB1 and λ5 from the λ5-IRES-VpreB1-EGFP retroviral vector was demonstrated by transducing the immature B cell lymphoma WEHI231 and staining for surface expression of total pre-BCR or λ5 (Figure 7B). Although the SL and endogenous kappa light chain (κLC) can compete for intracellular μHC, no reduction of surface κLC was detected in λ5-IRES-VpreB1-EGFP transduced WEHI231 cells, indicating an excess of endogenous intracellular μHC (Figure 7B). Next, IL-7 dependent pro/pre-B cell cultures were transduced with a construct expressing λ5-IRES-VpreB1-EGFP, or with a control construct expressing only EGFP (see experimental scheme in Figure 7C). Removal of IL-7 promotes maturation of the cultured pro/pre-B cells into immature B cells which express surface IgM [51] and κLC on their cell surface. After 5 to 7 days culture in the absence of IL-7, λ5-IRES-VpreB1-EGFP transduced cells produced only ∼50% of surface κLC+ cells compared to control transduced cells expressing only EGFP (Table 1). Hence, overexpression of the surrogate light chain interferes with the maturation of pro/pre-B cells to more mature κLC+ expressing B cells in vitro.


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)

Assessing the role of VpreB1 and λ5 expression for pro B cells maturation in vitro.(A) In the upper part a schematic of the retroviral construct used to transduce pro/pre-B cells is presented. The control construct is identical, but only contains EGFP. (B) WEHI231 lymphoma B cells were transduced with either VpreB1-IRES-λ5-EGFP or an EGFP retroviral expression construct. Expression of VpreB1 and λ5 was determined by staining for surface expression of the pre-BCR (SL156) and λ5 (LM34). Possible displacement of κLC on transduced WEHI231 cells was determined by staining for surface κLC. Representative stainings are presented. (C) Schematic of the transduction experiment with bone marrow cells. Hematopoietic stem cells (HSC) were first transduced with a Bcl-2 expressing retrovirus to enhance their survival and were differentiated into pro/preB cultures in the presence of Flt3 and IL-7. Subsequently, these cultures were transduced with the indicated retroviruses and differentiated in vitro by withdrawing IL-7.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003568-g007: Assessing the role of VpreB1 and λ5 expression for pro B cells maturation in vitro.(A) In the upper part a schematic of the retroviral construct used to transduce pro/pre-B cells is presented. The control construct is identical, but only contains EGFP. (B) WEHI231 lymphoma B cells were transduced with either VpreB1-IRES-λ5-EGFP or an EGFP retroviral expression construct. Expression of VpreB1 and λ5 was determined by staining for surface expression of the pre-BCR (SL156) and λ5 (LM34). Possible displacement of κLC on transduced WEHI231 cells was determined by staining for surface κLC. Representative stainings are presented. (C) Schematic of the transduction experiment with bone marrow cells. Hematopoietic stem cells (HSC) were first transduced with a Bcl-2 expressing retrovirus to enhance their survival and were differentiated into pro/preB cultures in the presence of Flt3 and IL-7. Subsequently, these cultures were transduced with the indicated retroviruses and differentiated in vitro by withdrawing IL-7.
Mentions: The expression of members of the surrogate light chain VpreB1, 2 and λ5 is tightly controlled at the transition from pre-BI, large and small pre-BII, to immature B cells, suggesting that this downregulation is essential. In order to test whether high levels of the surrogate light chain could interfere with the maturation of early B cell stages, we set out to overexpress λ5 and VpreB1 in early B cells, using a λ5-IRES-VpreB1-EGFP retroviral expression vector (Figure 7A). First, expression of VpreB1 and λ5 from the λ5-IRES-VpreB1-EGFP retroviral vector was demonstrated by transducing the immature B cell lymphoma WEHI231 and staining for surface expression of total pre-BCR or λ5 (Figure 7B). Although the SL and endogenous kappa light chain (κLC) can compete for intracellular μHC, no reduction of surface κLC was detected in λ5-IRES-VpreB1-EGFP transduced WEHI231 cells, indicating an excess of endogenous intracellular μHC (Figure 7B). Next, IL-7 dependent pro/pre-B cell cultures were transduced with a construct expressing λ5-IRES-VpreB1-EGFP, or with a control construct expressing only EGFP (see experimental scheme in Figure 7C). Removal of IL-7 promotes maturation of the cultured pro/pre-B cells into immature B cells which express surface IgM [51] and κLC on their cell surface. After 5 to 7 days culture in the absence of IL-7, λ5-IRES-VpreB1-EGFP transduced cells produced only ∼50% of surface κLC+ cells compared to control transduced cells expressing only EGFP (Table 1). Hence, overexpression of the surrogate light chain interferes with the maturation of pro/pre-B cells to more mature κLC+ expressing B cells in vitro.

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