Dominant-Negative Impact of PAX5/TEL on Downstream Targets of PAX5 and Essential Pre-B Cell Receptor Genes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3231-3231
Author(s):  
Gabriela B. Iwanski ◽  
Nils H. Thoennissen ◽  
Joy Nakitandwe ◽  
Patricia Lin ◽  
Norihiko Kawamata ◽  
...  
Keyword(s):  
B Cell ◽  
Target Genes ◽  
Fusion Gene ◽  
Negative Impact ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Dominant Negative ◽  
Bcr Signaling ◽  
Pediatric All

Abstract Abstract 3231 B cell lineage acute lymphoblastic leukemia (ALL) is a common malignancy in childhood, and the pre-B cell receptor (pre-BCR) signalling pathway was previously demonstrated to function as a tumor suppressor. The transcription factor PAX5, a key regulator of B cell development, is frequently involved in chromosomal rearrangements of leukemic blasts. Using high resolution single nucleotide polymorphism (SNP) genomic microarray by us and other groups, several candidate partner genes fused to PAX5 have been detected in samples of pediatric ALL, such as TEL, FOXP1, AUTS2, and C20orf112. Recently, we studied the fusion gene PAX5/TEL and its role in leukemic evolution by Affymetrix HG-U133 plus 2.0 Array of the ALL cell line Nalm6 transfected with a PAX5/TEL construct. PAX5/TEL reduced the expression of PAX5 and its downstream target genes (e.g. CD79A, BACH2, CD19). Moreover, we demonstrated a dominant negative impact of the PAX5/TEL-fusion protein on the binding affinity of wild-type PAX5 to the promoter of CD79A (Iwanski et al., 2009 ASH Abstract No. 3455). To expand our findings, we analyzed the gene expression profile of pediatric ALL samples carrying PAX5/TEL (PAX5/TEL+, n=2) compared to samples with normal PAX5 (n=7) from a genomic ALL study. Samples with normal PAX5 were selected from among 95 B-ALL patients with normal PAX5 status, based on characteristics that most closely matched the two PAX5-TEL+ patients including cytogenetics and current risk stratification. Gene expression data were compiled using the Affymetrix HG-U133A Array, and a heatmap based on the Top 200 probes with the highest expression levels from both sample sets was generated (TIBCO Software Inc.). Notably, the downregulated genes included Bruton agammaglobulinemia tyrosine kinase (BTK; -2.8 fold, FDR < 0.2), an important regulator of pre-BCR signaling, Spleen tyrosine kinase (SYK, -2.3 fold, FDR < 0.2), and IGHM (-5,9 fold; FDR < 0.1), but also significantly up-regulated expression of genes involved in myeloid differentiation, namely Myeloperoxidase (MPO, +24.2 fold, FDR < 0.2), and CCAAT/enhancer binding protein alpha (CEBPA, +3.2 fold, FDR < 0.3), as well as the erythroid genes Aminolevulinate delta-dehydratase (ALAD, +12.2 fold, FDR < 0.2) and the Erythropoietin receptor (EPOR, +7.0, FDR < 0.3). Additionally, we performed a meta-analysis comparing deregulated genes detected in the Nalm6-microarray (PAX5/TEL-MIGR vs. empty vector) to the data from the human B-ALL samples (PAX5/TEL+ vs. normal PAX5). Overall, we identified a set of 35 overlapping genes (FDR < 0.3) that were deregulated in both data sets (21 downregulated, 14 up-regulated). Notably, 10 out of the 21 (47%) downregulated genes are known to be involved in B cell development and BCR signaling, some of them well-recognized as direct PAX5 target genes (e.g. CD79A, CD19, BACH2). Moreover, reporter gene assay with a luciferase reporter construct containing cDNA of the CD19 promoter with PAX5 binding sites (luc-CD19) was performed in Nalm6 cells. Since these cells already express a high level of endogenous PAX5, transcriptional activity of the luc-CD19 reporter plasmid was relatively high in the Nalm6 cells transfected with empty vector, as compared to 293T cells. In contrast, PAX5/TEL-transduced Nalm6 cells displayed a significantly reduced transcriptional activation of the reporter construct (P < 0.01). We also explored if mutation and/or deletion of PAX5 (mut/del PAX5) may have an impact on genes involved in B cell development and the pre-BCR/BCR pathway. Hence, expression files from human ALL samples with mut/del PAX5 (n=50) and B-ALL samples with normal PAX5 (n=95) were analyzed. Notably, only two genes that are known to be involved in B cell development and the pre-BCR pathway were significantly downregulated in samples with mut/del PAX5 compared to normal PAX5, namely CD72, a B cell specific repressor gene activated by PAX5 (-1.51 mean fold, FDR = 0.05), and immunoglobulin heavy constant delta (IGHD), a gene involved in pre-BCR signalling (-1.6 mean fold, FDR = 0.18). These findings suggest no strong influence of mut/del PAX5 on the expression of downstream genes involved in pre-BCR signaling. In conclusion, our results provide further insights into the dominant-negative role of PAX5/TEL and link this fusion gene with the pre-BCR pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals KZF-L162R Mutation Affects Splenic Mature B Cell Development and Alters Expression of Aiolos Target Genes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 668-668
Author(s):  
Gregory Lazarian ◽  
Shanye Yin ◽  
Alba Font-tello ◽  
Elisa Ten Hacken ◽  
Tomasz Sevastianik ◽  
...  
Keyword(s):  
B Cells ◽  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Cell Development ◽  
B Cell Development ◽  
Wild Type ◽  
Bcr Signaling ◽  
Average Proportion ◽  
Follicular B Cells

Abstract Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL. To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT). Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3WTor Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161RHetmice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHetmice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161RHomomice are ongoing. By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161RHetmice (9 mice/group; p<0.05). Since the mutation localizes to a DNA binding domain, we hypothesized that it modifies the ability of Aiolos to control expression of its target genes. We therefore performed CHIP-seq in Ikzf3WTsplenic B cells, and identified Aiolos-associated high confidence peaks (fold change (FC) enrichment compared to input > 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC>10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos. Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161RHomoB cells (p<0.05) but not in Ikzf3-L161RHetB cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease. In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner. Disclosures Wu: Neon Therapeutics: Equity Ownership.

scholarly journals Distinct Roles of Phosphoinositide-3 Kinase and Phospholipase Cγ2 in B-Cell Receptor-Mediated Signal Transduction

2006 ◽  
Vol 26 (1) ◽  
pp. 88-99 ◽  
Author(s):  
Xuezhi Dai ◽  
Yuhong Chen ◽  
James Schuman ◽  
Zichun Hua ◽  
John W. Adamson ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
Phosphoinositide 3 Kinase ◽  
Cell Transition

ABSTRACT During B-cell receptor (BCR) signaling, phosphoinositide-3 kinase (PI3K) is thought to function upstream of phospholipase Cγ2 (PLCγ2). PLCγ2 deficiency specifically impedes transitional type 2 (T2) to follicular (FO) mature B-cell transition. Here, we demonstrate that PI3K deficiency specifically impaired T2-to-FO mature B-cell transition and marginal zone B-cell development. Furthermore, we investigated the functional relationship between PI3K and PLCγ2 using PI3K−/−, PLCγ2−/−, and PI3K−/− PLCγ2−/− B cells. Interestingly, PLCγ2 deficiency had no effect on BCR-mediated PI3K activation, whereas PI3K deficiency only partially blocked activation of PLCγ2. Moreover, whereas PI3K−/− PLCγ2−/− double deficiency did not affect hematopoiesis, it resulted in embryonic lethality. PI3K−/− PLCγ2−/− fetal liver cells transplanted into B-cell null JAK3−/− mice failed to restore development of peripheral B cells and failed to progress through early B-cell development at the pro-B- to pre-B-cell transition, a more severe phenotype than was observed with either PI3K or PLCγ2 single-deficiency B cells. Consistent with this finding, BCR signaling was more severely impaired in the absence of both PI3K and PLCγ2 genes than in the absence of either one alone. Taken together, these results demonstrate that whereas PI3K functions upstream of PLCγ2, activation of PLCγ2 can occur independently of PI3K and that PI3K and PLCγ2 also have distinct functions in BCR signal transduction.

scholarly journals WASP and Mst1 coregulate B-cell development and B-cell receptor signaling

2020 ◽  
Vol 4 (3) ◽  
pp. 573-585 ◽  
Author(s):  
Lu Huang ◽  
Xiaoyu Sun ◽  
Di Yang ◽  
Xin Dai ◽  
Panpan Jiang ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Actin Dynamics ◽  
Double Knockout ◽  
Bcr Signaling

Abstract Mst1 is a serine/threonine kinase involved in cell survival, proliferation, apoptosis, and tumorigenesis. In mice, Mst1 regulates actin dynamics required for T-cell adhesion and migration, which correlate with thymic egress and entry into lymphatic tissue. The role of Mst1 in B cells and how it may control actin-dependent processes has not been well characterized. Wiskott-Aldrich syndrome protein (WASP) deficiency only moderately affects development and B-cell receptor (BCR) signaling, suggesting WASP likely associates with other molecules. We investigated whether Mst1 associates with WASP to regulate B-cell development and activation. Experimenting on Mst1/WASP double knockout (DKO) mice, we found a severe defect in the bone marrow B-cell development, and BCR signaling in the DKO mice was severely reduced. Even though WASP or Mst1 could influence the early B-cell activation, we found that the early activation events such as B-cell spreading, BCR clustering, and BCR signaling were much more impaired in the B cells from DKO mice. Furthermore, reciprocal regulation between Mst1 and WASP was observed in WASP and Mst1 KO mice, whereby the localization and function of phosphorylated WASP were affected in Mst1 KO mice. Most importantly, Mst1 inhibits the expression of WASP by decreasing the expression of WASP-interacting protein. Interestingly, we also found that WASP deficiency in patients and mice interferes with phosphorylated Mst1 localization and therefore function in B cells. Overall, our study provides a partner for WASP to regulate B-cell development and BCR signaling, as well as the reciprocal regulating molecular mechanism of one another.

scholarly journals IL-7 receptor signaling is necessary for stage transition in adult B cell development through up-regulation of EBF

2005 ◽  
Vol 201 (8) ◽  
pp. 1197-1203 ◽  
Author(s):  
Kazu Kikuchi ◽  
Anne Y. Lai ◽  
Chia-Lin Hsu ◽  
Motonari Kondo
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
B Cell ◽  
Target Genes ◽  
Cell Development ◽  
B Cell Development ◽  
Receptor Signaling ◽  
Cell Stage ◽  
Transcription Factor Network ◽  
Stage Transition

Cytokine receptor signals have been suggested to stimulate cell differentiation during hemato/lymphopoiesis. Such action, however, has not been clearly demonstrated. Here, we show that adult B cell development in IL-7−/− and IL-7Rα2/− mice is arrested at the pre–pro-B cell stage due to insufficient expression of the B cell–specific transcription factor EBF and its target genes, which form a transcription factor network in determining B lineage specification. EBF expression is restored in IL-7−/− pre–pro-B cells upon IL-7 stimulation or in IL-7Rα−/− pre–pro-B cells by activation of STAT5, a major signaling molecule downstream of the IL-7R signaling pathway. Furthermore, enforced EBF expression partially rescues B cell development in IL-7Rα−/− mice. Thus, IL-7 receptor signaling is a participant in the formation of the transcription factor network during B lymphopoiesis by up-regulating EBF, allowing stage transition from the pre–pro-B to further maturational stages.

scholarly journals Secreted IgM deficiency leads to increased BCR signaling that results in abnormal splenic B cell development

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Dimitrios Tsiantoulas ◽  
Mate Kiss ◽  
Barbara Bartolini-Gritti ◽  
Andreas Bergthaler ◽  
Ziad Mallat ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Bcr Signaling ◽  
Igm Deficiency

scholarly journals Functions of E2A-HEB Heterodimers in T-Cell Development Revealed by a Dominant Negative Mutation of HEB

2000 ◽  
Vol 20 (18) ◽  
pp. 6677-6685 ◽  
Author(s):  
Robert J. Barndt ◽  
Meifang Dai ◽  
Yuan Zhuang
Keyword(s):  
T Cell ◽  
Target Genes ◽  
T Cell Development ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Cell Development ◽  
Dominant Negative ◽  
Helix Loop Helix ◽  
Dominant Negative Mutation ◽  
Bhlh Proteins

ABSTRACT Lymphocyte development and differentiation are regulated by the basic helix-loop-helix (bHLH) transcription factors encoded by theE2A and HEB genes. These bHLH proteins bind to E-box enhancers in the form of homodimers or heterodimers and, consequently, activate transcription of the target genes. E2A homodimers are the predominant bHLH proteins present in B-lineage cells and are shown genetically to play critical roles in B-cell development. E2A-HEB heterodimers, the major bHLH dimers found in thymocyte extracts, are thought to play a similar role in T-cell development. However, disruption of either the E2A or HEBgene led to only partial blocks in T-cell development. The exact role of E2A-HEB heterodimers and possibly the E2A and HEB homodimers in T-cell development cannot be distinguished in simple disruption analysis due to a functional compensation from the residual bHLH homodimers. To further define the function of E2A-HEB heterodimers, we generated and analyzed a dominant negative allele of HEB, which produces a physiological amount of HEB proteins capable of forming nonfunctional heterodimers with E2A proteins. Mice carrying this mutation show a stronger and earlier block in T-cell development than HEB complete knockout mice. The developmental block is specific to the α/β T-cell lineage at a stage before the completion of V(D)J recombination at the TCRβ gene locus. This defect is intrinsic to the T-cell lineage and cannot be rescued by expression of a functional T-cell receptor transgene. These results indicate that E2A-HEB heterodimers play obligatory roles both before and after TCRβ gene rearrangement during the α/β lineage T-cell development.

#630 Role of the pre-B cell receptor complex in B cell development

1998 ◽  
Vol 20 (4) ◽  
pp. 383
Author(s):  
S. R. Rheingold ◽  
M. Jiang ◽  
S. A. Grupp ◽  
B. Himelstein
Keyword(s):  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Receptor Complex

scholarly journals Essential Immunoregulatory Role for BCAP in B Cell Development and Function

2002 ◽  
Vol 195 (5) ◽  
pp. 535-545 ◽  
Author(s):  
Tetsuo Yamazaki ◽  
Kiyoshi Takeda ◽  
Kumiko Gotoh ◽  
Hiroshi Takeshima ◽  
Shizuo Akira ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Phosphoinositide 3 Kinase ◽  
And Function ◽  
B Cell Deficiency

BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell–independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca2+ mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

Identification of Cyclin D3 as a Direct Transcriptional Target of E2A Using Damid.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1618-1618
Author(s):  
John K. Choi ◽  
Siyuan Song ◽  
Jonathan Cooperman ◽  
Danielle L. Letting ◽  
Gerd A. Blobel
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Cell Development ◽  
Binding Activity ◽  
B Cell Development ◽  
Cyclin D3 ◽  
Dna Binding Activity ◽  
E Boxes

Abstract The transcription factor E2A is required for very early B cell development. The exact mechanism by which E2A promotes B cell development is unclear and cannot be explained by the known E2A targets, components of the pre-B cell receptor and cyclin dependent kinase inhibitors, indicating additional pathways and targets remain to be identified. We had previously reported that E2A can promote precursor B cell expansion, promote G1 cell cycle progression, and induce the expressions of multiple G1 phase cyclins including cyclin D3, suggesting that E2A induction of these genes may contribute to early B cell development. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated a luciferase reporter plasmid containing the 1kb promoter of cyclin D3 that contains two consensus E2A binding sites (E-boxes); however, deletion of the E-boxes did not disrupt the transactivation by E2A. We hypothesized three possible mechanisms: 1) indirect activation of cyclin D3 via another transcription factor, 2) binding of E2A to cryptic non-E-boxes, or 3) recruitment of E2A to the promoter via interaction with other DNA binding factor. To test the first possibility, promoter occupancy was examined using the DamID approach. In this approach, a fusion protein consisting of E. coli DNA adenosine methyltransferase (DAM) and a transcription factor of interest is expressed at low levels, resulting in specific methylation of adenosine residues within 2–5 kb of the transcription factor target sites. A fusion construct composed of E2A and DAM (E47Dam), was subcloned in lentiviral vectors, and used to transduce precursor B cell lines. The methylated adenosine residues were detected using a sensitive ligation-mediated PCR (LM-PCR) assay that required only 1 ug of genomic DNA and can detect methylation even if only 3% of the cells express E47Dam; no methylated adenosines were detected in control cells, indicating that all methylated residues resulted from E47Dam. Specific adenosine methylation was identified at the IgH intronic enhancer, a known E2A target site, but not at the non-target sites, CD19, HPRT, and GAPDH promoters. Specific methylation was detected at the cyclin D3 promoter but not 10 kb down-stream, despite similar concentrations of E-boxes at both sites. Chromatin immunoprecipitation analysis confirmed the DamID findings and further localized the binding to within 1 kb of the two E-boxes in the cyclin D3 promoter. To distinguish between the two remaining mechanisms (cryptic non-E-boxes versus recruitment via other DNA binding factors), two point mutations were introduced into E47Dam that disrupted its DNA binding activity. The mutated E47Dam continued to methylate at the cyclin D3 promoter. We conclude that E2A can be recruited to the cyclin D3 promoter, independent of E-boxes or E2A DNA binding activity. Our findings raise the possibility that some direct E2A target genes may lack functional E-boxes. Furthermore, mutated E2A, lacking an E2A DNA binding domain, that is seen in 6% of pediatric ALLs may still activate a subset of E2A target genes. Finally, our application of lentiviral vectors and LM-PCR to the DamID approach should permit analysis of primary human precursor B cells, despite the limitations in cell number and transduction efficiency.

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