Down-regulation of RAG1 and RAG2 gene expression in PreB cells after functional immunoglobulin heavy chain rearrangement

Grawunder, Ulf; Leu, Thomas; Schatz, David G.; Werner, Annick; Rolink, Antonius; Melchers, Fritz; Winkler, Thomas

doi:10.1016/1074-7613(95)90131-0

Cited by 332 publications

(274 citation statements)

References 33 publications

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“…Large pre-BI as well as pre-BII cells are presumably in S/G2/M, while small cells are in G0/G1 of the cell cycle [37,38]. Our results show that large cells express higher levels of the IL-7R than small cells.…”

Section: Resultsmentioning

confidence: 52%

“…Independent of genotype, large cells expressed higher levels than small cells (MFI of *19 compared to *7). The percentages of both large (63% and 81%, respectively) and small (20% and 31%, respectively) pre-BII cells expressing the IL-7R were lower in VpreB -/-compared to control mice.Large pre-BI as well as pre-BII cells are presumably in S/G2/M, while small cells are in G0/G1 of the cell cycle [37,38]. Our results show that large cells express higher levels of the IL-7R than small cells.…”

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Both the pre-BCR and the IL-7Rα are essential for expansion at the pre-BII cell stagein vivo

et al. 2005

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During B cell development, proliferative expansion takes place after expression of the pre-BCR. At this pre-BII cell stage, the IL-7Ra is also expressed. Some in vitro studies suggest that pre-BCR-dependent expansion relies on the IL-7Ra, and others that it does not. It has also been suggested that the pre-BCR mediates down-regulation of the IL7Ra. However, the in vivo relationship between the pre-BCR and the IL-7Ra has not been previously examined. Here, we have investigated this by establishing mice lacking both receptors. Our results show that in the absence of the IL-7Ra, the pre-BII population is reduced, as previously seen in mice lacking the pre-BCR, demonstrating that the IL-7Ra is important at this stage. A deficiency in both receptors results in a further reduction of the pre-BII cell population. We conclude that both the IL-7Ra and the pre-BCR are required for optimal pre-BII cell expansion. Furthermore, IL-7Ra expression levels are normal in pre-BCR-deficient mice, suggesting that the pre-BCR does not mediate its down-regulation. As a consequence of the absence of both receptors, the peripheral B cell pool is severely depleted, resulting in atypical splenic B cell structures and reduced serum Ig levels. IntroductionB lymphopoiesis takes place in the fetal liver and, postnatally, in the BM [1][2][3]. During early BM B cell development, critical checkpoints occur firstly after Ig heavy (H)-chain DJ H rearrangement and secondly after successful VDJ H rearrangement. Due to the inherent imprecision of the rearrangement process, each of these is followed by proliferative expansion at the pre-BI (DJ H ) and large pre-BII (VDJ H ) cell stages, which results in an increase in cell numbers and, consequently, the Ig repertoire. Functional VDJ H -rearrangement leads to expression of the lH chain, which, together with the surrogate light chain (SLC, comprised of VpreB and k5) [4, 5], forms the pre-B cell receptor (pre-BCR) [6][7][8]. After subsequent Ig light (L)-chain recombination, although not followed by an expansion phase, the end result is the production of large numbers of immature B cells, each carrying a unique BCR (IgM) on the cell surface. Immature B cells migrate to the spleen, where they mature and co-express IgD [9].Expansion at the pre-BI cell stage has been shown, both in vitro [10][11][12][13] and in vivo [14][15][16][17][18][19][20][21], to depend on the IL-7Ra [22]. Mice deficient in the IL-7Ra chain show a block in B cell development at the pre-BI stage. Although this block is complete in adult IL-7Ra -/-mice, Leukocyte signalingThe last two authors contributed equally to this work. [20,21]. The IL-7Ra chain heterodimerizes with the common gamma (c C ) chain [22,23] to form the receptor for IL-7, while together with the thymic stromal lymphopoietin (TSLP) receptor chain [24,25], it forms the receptor for TSLP. Expansion at the pre-BII stage is dependent on the expression of the pre-BCR. While a lack of the transmembrane region of the lH chain (lMT) leads to a complete block at the pre-BI stag...

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Section: Resultsmentioning

confidence: 52%

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confidence: 52%

Both the pre-BCR and the IL-7Rα are essential for expansion at the pre-BII cell stagein vivo

et al. 2005

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“…Oncogene (2006Oncogene ( ) 25, 5180-5186. doi:10.1038published online 24 April 2006 Keywords: V(D)J recombination; pre-B cell receptor; leukemia; SH2-domain; DNA rearrangement Perpetual V(D)J recombinase activity continuously generates DNA double-strand breaks and may give rise to secondary transforming events during the malignant progression of early leukemia and lymphoma cells (Khanna and Jackson, 2001). In B-cell precursors, V(D)J recombination is regulated through a negative feedback signal: upon successful rearrangement, a m-heavy chain encoded by a productively rearranged V H region gene signals termination of recombination activity at the IGHV locus (Grawunder et al, 1995). How this negative feedback signal is deranged in leukemia and lymphoma cells is not yet resolved.…”

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confidence: 99%

SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells

et al. 2006

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Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-Blymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V H gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V H -DJ H rearrangements and secondary V H replacement. We conclude that iterative V H gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.Oncogene ( Recent work demonstrated that deficiency of SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) is a frequent feature in acute lymphoblastic leukemia cells (Jumaa et al., 2003;Klein et al., 2004). Although a recent report questioned these findings (Imai et al., 2004), this study shows that defective SLP65 expression is not only frequent in human pre-B-lymphoblastic leukemia but also occurs in a fraction of mature B-cell lymphoma cases. Identifying three leukemia and one lymphoma cell line lacking expression of functional SLP65, we studied the contribution of SLP65 to the control of the V(D)J recombinase activity in B-cell lineage leukemia and lymphoma cells. Perpetual V(D)J recombinase activity in B-cell lineage leukemia and lymphoma cellsIn order to investigate ongoing V(D)J recombinase activity in B-cell precursor leukemia and B-cell lymphoma cells, we first analysed the configuration of immunoglobulin (Ig) gene loci in leukemia and lymphoma cell lines. Among 22 clonal pre-B-lymphoblastic leukemia and B-cell lymphoma cell lines, five of 12 pre-Blymphoblastic leukemia and two of 10 B-cell lymphoma cell lines express RAG1 and RAG2, and carry more than two productively rearranged Ig heavy chain V region genes, indicating that negative feedback signaling of the (pre-) B-cell receptor to V(D)J recombinase activity was impaired in these cells (Table 1). In (pre-) B-lymphoblastic cell lines harboring only one productively rearranged IGHV allele, expression of RAG1 and RAG2 does not necessarily indicate defective negative feedback signaling of the (pre-) B-cell receptor and may also reflect active rearrangement of IGKV and IGLV light chain genes. In addition, ongoing V(D)J recombinase activity represents a typical feature of pre-B-lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement as previously shown by us and others (Height et al., 1996;Klein et al., 2004), suggestin...

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“…RAG proteins act together to recognize recombination signal sequences that flank V, D, and J gene segments and introduce the double-stranded DNA breaks between the recombination signal sequences and the gene segments [3]. The expression of RAG-1 and RAG-2 is restricted to lymphoid cells undergoing V(D)J rearrangement [4, 5]. In the absence of either RAG-1 or RAG-2 gene products, the development of mature lymphocytes is completely abrogated and hence results in severe combined immune deficiency in mouse and human [6][7][8].…”

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“…2C). It has been reported that RAG-2 is not expressed naturally in mature lymphocytes [4,10]. The abnormal expression maybe implies that Ep needs to associate with an additional flanking region, such as a matrix attachment region, for its correct regulation in vivo [27,28].…”

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Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene‐2

et al. 2005

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Recombination-activating gene (RAG)-1 and RAG-2 are essential for V(D)J recombination and are expressed specifically in lymphoid cells. We previously identified two putative enhancer elements, the proximal and distal enhancers, located at -2.6 and -8 kb, respectively, 5 0 upstream of mouse RAG-2, and characterized the distal enhancer element in detail. In this study, to characterize the proximal enhancer in vitro as well as in vivo, we first defined a 170-bp core enhancer element within the proximal enhancer (Ep) and determined its activity in various cells. Ep conferred enhancer activity only in B-lymphoid cell lines, but not in T-or non-lymphoid cell lines. Analysis of the transgenic mice carrying an EGFP reporter gene linked with Ep revealed that Ep activated the transcription of the reporter gene in bone marrow and spleen, but not in thymus or non-lymphoid tissues. Ep was active in both B220 + IgM -and B220 + IgM + subpopulations in the bone marrow and in the B220 + subpopulation in the spleen. Using electrophoretic mobility shift assays and mutational assays, we found that Ikaros and CCAAT/enhancer binding protein cooperatively bind Ep and function as the transcription factors responsible for B cell-specific enhancer activity. These results demonstrate the role of Ep as a cis-regulatory enhancer element for RAG-2-specific expression in B-lymphoid lineages. IntroductionAssembly of Ig and TCR genes by V(D)J recombination is mediated by the products of recombination-activating gene (RAG)-1 and RAG-2 [1, 2]. RAG proteins act together to recognize recombination signal sequences that flank V, D, and J gene segments and introduce the double-stranded DNA breaks between the recombination signal sequences and the gene segments [3]. The expression of RAG-1 and RAG-2 is restricted to lymphoid cells undergoing V(D)J rearrangement [4, 5]. In the absence of either RAG-1 or RAG-2 gene products, the development of mature lymphocytes is completely abrogated and hence results in severe combined immune deficiency in mouse and human [6][7][8]. In human, missense mutations in either RAG-1 or RAG-2 result in the Omenn syndrome, which is characterized by a poor T lymphocyte repertoire [9].There are two waves of RAG-1 and RAG-2 expression in bone marrow B cell precursors. RAG are expressed first in pro-B cell progenitors undergoing Ig heavy-chain gene rearrangement, and the second expression of RAG is seen during Ig j or k light-chain gene rearrangement occurring in pre-B cells [4,10]. In T cell development, RAG areThe first two authors contributed to this work equally. first expressed in pro-T cells when rearrangement of the TCRb chain gene occurs, and secondary expression of RAG occurs when the TCRa chain loci undergo V-J rearrangement to produce mature T cells [11,12]. The transcription of RAG is regulated at different levels. At the chromatin level, Fuller et al. and we reported that a DNase I-hypersensitive (HS) site was identified in the promoter region of mouse and human RAG-1 only in RAG-expressing lymphocytes [13,14]. We a...

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Down-regulation of RAG1 and RAG2 gene expression in PreB cells after functional immunoglobulin heavy chain rearrangement

Cited by 332 publications

References 33 publications

Both the pre-BCR and the IL-7Rα are essential for expansion at the pre-BII cell stagein vivo

Both the pre-BCR and the IL-7Rα are essential for expansion at the pre-BII cell stagein vivo

SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells

Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene‐2

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