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Meinrad Busslinger - One of the best experts on this subject based on the ideXlab platform.

  • gabaergic neurons regulate lateral ventricular development via Transcription Factor PAX5
    Genesis, 2013
    Co-Authors: Nobuhisa Ohtsuka, Sylvia Badurek, Meinrad Busslinger, Francine M Benes, Liliana Minichiello, Uwe Rudolph
    Abstract:

    Summary Postmortem studies have revealed a downregulation of the Transcription Factor PAX5 in GABAergic neurons in bipolar disorder, a neurodevelopmental disorder, raising the question whether PAX5 in GABAergic neurons has a role in normal brain development. In a genetic approach to study functions of PAX5 in GABAergic neurons, PAX5 was specifically deleted in GABAergic neurons from PAX5 floxed mice using a novel Gad1-Cre transgenic mouse line expressing Cre recombinase in Gad1-positive, that is, GABAergic neurons. Surprisingly, these mice developed a marked enlargement of the lateral ventricles at approximately 7 weeks of age, which was lethal within 1–2 weeks of its appearance. This hydrocephalus phenotype was observed in mice homozygous or heterozygous for the PAX5 conditional knockout, with a gene dosage-dependent penetrance. By QTL (quantitative trait loci) mapping, a 3.5 Mb segment on mouse chromosome 4 flanked by markers D4Mit237 and D4Mit214 containing approximately 92 genes including PAX5 has previously been linked to differences in lateral ventricular size. Our findings are consistent with PAX5 being a relevant gene underlying this QTL phenotype and demonstrate that PAX5 in GABAergic neurons is essential for normal ventricular development. genesis 51:234–245. © 2013 Wiley Periodicals, Inc.

  • the Transcription Factor PAX5 regulates its target genes by recruiting chromatin modifying proteins in committed b cells
    The EMBO Journal, 2011
    Co-Authors: Shane Mcmanus, Giorgia Salvagiotto, Hiromi Tagoh, Anja Ebert, Jasna Medvedovic, Ido Tamir, Markus Jaritz, Meinrad Busslinger
    Abstract:

    PAX5 is a critical regulator of B-cell commitment. Here, we identified direct PAX5 target genes by streptavidin-mediated ChIP-chip analysis of pro-B cells expressing in vivo biotinylated PAX5. By binding to promoters and enhancers, PAX5 directly regulates the expression of multiple Transcription Factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer PAX5-dependent B-cell-specific activity to the Nedd9 gene controlling B-cell trafficking. Profiling of histone modifications in PAX5-deficient and wild-type pro-B cells demonstrated that PAX5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro-B cells. PAX5 rapidly induces these chromatin and Transcription changes by recruiting chromatin-remodelling, histone-modifying and basal Transcription Factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which PAX5 controls B-cell commitment.

  • Transcription Factor PAX5 activates the chromatin of key genes involved in b cell signaling adhesion migration and immune function
    Immunity, 2007
    Co-Authors: Alexandra Schebesta, Shane Mcmanus, Giorgia Salvagiotto, Alessio Delogu, Georg A Busslinger, Meinrad Busslinger
    Abstract:

    Summary The Transcription Factor PAX5 represses B lineage-inappropriate genes and activates B cell-specific genes in B lymphocytes. Here we have identified 170 PAX5-activated genes. Conditional mutagenesis demonstrated that the PAX5-regulated genes require continuous PAX5 activity for normal expression in pro-B and mature B cells. Expression of half of the PAX5-activated genes is either absent or substantially reduced upon PAX5 loss in plasma cells. Direct PAX5 target genes were identified based on their protein synthesis-independent activation by a PAX5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of PAX5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that PAX5 directly activates the chromatin at promoters or putative enhancers of PAX5 target genes. The PAX5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation, and germinal-center B cell formation, thus revealing a complex regulatory network that is activated by PAX5 to control B cell development and function.

  • Reporter gene insertions reveal a strictly B lymphoid-specific expression pattern of PAX5 in support of its B cell identity function.
    Journal of immunology (Baltimore Md. : 1950), 2007
    Co-Authors: Martin Fuxa, Meinrad Busslinger
    Abstract:

    The Transcription Factor PAX5 is essential for B cell commitment and development. Although the detail PAX5 expression pattern within the hemopoietic system is still largely unknown, we previously reported that PAX5 is monoallelically transcribed in pro-B and mature B cells. In this study, we have investigated the expression of PAX5 at single-cell resolution by inserting a GFP or human Cd2 indicator gene under the translational control of an internal ribosomal entry sequence into the 3' untranslated region of PAX5. These insertions were noninvasive, as B cell development was normal in PAX5(ihCd2/ihCd2) and PAX5(ihGFP/iGFP) mice. Transheterozygous PAX5(ihCd2/iGFP) mice coexpressed GPF and human CD2 at similar levels from pro-B to mature B cells, thus demonstrating biallelic expression of PAX5 at all stages of B cell development. No reporter gene expression could be detected in plasma cells and non-B cells of hemopoietic system. Moreover, the vast majority of common lymphoid progenitors and pre-pro-B in the bone marrow of PAX5(ihGFP/iGFP) mice did not yet express GFP, indicating that PAX5 expression is fully switched on only during the transition form uncommitted pre-pro-B cells to committed pro-B cells. Hence, the Transcriptional initiation and B cell-specific expression of PAX5 is entirely consistent with its B cell lineage commitment function.

  • PAX5: the guardian of B cell identity and function
    Nature Immunology, 2007
    Co-Authors: César Cobaleda, Alessio Delogu, Alexandra Schebesta, Meinrad Busslinger
    Abstract:

    The Transcription Factor PAX5 is essential for commitment of lymphoid progenitors to the B lymphocyte lineage. PAX5 fulfils a dual role by repressing B lineage 'inappropriate' genes and simultaneously activating B lineage–specific genes. This Transcriptional reprogramming restricts the broad signaling capacity of uncommitted progenitors to the B cell pathway, regulates cell adhesion and migration, induces V_H-DJ_H recombination, facilitates (pre-)B cell receptor signaling and promotes development to the mature B cell stage. Conditional PAX5 inactivation in early and late B lymphocytes revealed an essential role for PAX5 in controlling the identity and function of B cells throughout B lymphopoiesis. PAX5 has also been implicated in human B cell malignancies, as it is deregulated by chromosomal translocations in a subset of acute lymphoblastic leukemias and non-Hodgkin lymphomas.

James R Downing - One of the best experts on this subject based on the ideXlab platform.

  • Retroviral and Chemical Mutagenesis Identifies PAX5 as a Tumor Suppressor in B-Progenitor Acute Lymphoblastic Leukemia
    Blood, 2008
    Co-Authors: Jinjun Dang, Charles G Mullighan, Letha A. Phillips, Perdeep K. Mehta, James R Downing
    Abstract:

    Recent genome-wide analyses of acute lymphoblastic leukemia (ALL) have identified genetic alterations targeting the B lymphoid Transcription Factor PAX5 in over 30% of B-progenitor ALL (Nature 2007;446:758; Nature 2008;453:110). The PAX5 mutations include deletions, focal internal amplification, sequence mutations and translocations that result in PAX5 haploinsufficiency or generate PAX5 mutants with impaired DNA-binding or transactivating activitiy. In almost all B-ALL cases, the mutations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor in B-ALL. To test this hypothesis, we have performed mutagenesis screens of mice heterozygous for a PAX5 null allele ( PAX5 +/−; Cell 1994;79:901). In a chemical mutagenesis screen, thymectomized C57BL6/Sv129 PAX5 +/− (N=25) and wild type ( PAX5 +/+; N=20) mice received a single 100mg/kg dose of the alkylating agent N-ethyl-S-nitrosourea (ENU) at 4–5 weeks of age. PAX5 +/− animals exhibited a markedly increased frequency of leukemia in comparison to wild type animals. After one year, 24 of 25 PAX5 +/ − animals developed leukemia (median latency 246 days), compared to 3 of 20 PAX5 +/+ animals ( P

  • mutations in the b cell Transcription Factor PAX5 found in b progenitor acute lymphoblastic leukemia impair normal PAX5 activity
    Blood, 2006
    Co-Authors: Christopher B Miller, Charles G Mullighan, James R Downing
    Abstract:

    Using genome-wide profiling of DNA copy number abnormalities using high-resolution single nucleotide polymorphism arrays, we recently identified a high frequency of genomic aberrations involving the PAX5 gene in pediatric B-progenitor ALL. PAX5 is a critical Transcriptional regulator of B lymphocyte commitment and differentiation. Mutations, including partial tandem duplication, complete and focal deletions, point mutations in the DNA-binding or transactivation domain, and three translocations that encode PAX5 fusion proteins were observed in 31.7% of B-ALL. The PAX5 deletions were mono-allelic and resulted in either loss of the entire gene, or the deletion of only a subset of the exons leading to the production of PAX5 proteins that lacked the DNA-binding paired domain (exons 2–4) and/or the Transcriptional activation domain (exons 7–10). In murine systems, the complete absence of PAX5 results in the arrest of B-cell development at the pro-B-cell stage prior to immunoglobulin heavy chain rearrangement, whereas haploinsufficiency leads to a partial block in B-cell development. Importantly, in the primary leukemia samples, the mono-allelic loss of PAX5 was associated with reduced expression of PAX5 by flow cytometry and quantitative RT-PCR, suggesting that haploinsufficiency contributes to the block in differentiation characteristic of B-progenitor ALL. To determine if the other identified PAX5 mutations result in hypomorphic alleles, we analyzed the DNA-binding and Transcriptional activity of the encoded proteins. DNA-binding activity was assessed by electrophoretic mobility gel-shift assays using a labeled oligonucleotide probes from the promoters of the PAX5 target genes CD19 and CD79A (mb-1), and Transcriptional activity was assessed by a luciferase-based reporter assays using the PAX5-dependent reporter plasmid, luc -CD19. Analysis was performed on the paired-domain mutants P80R and P34Q, the focal deletions Δe2-5, Δe2-6, Δe2-7, Δe2-8, and Δe6-8, and the PAX5-ETV6 and PAX5-FOXP1 translocation-encoded fusion proteins. As expected, DNA-binding was abrogated in deletion mutants that lacked the paired domain (Δe2-5, Δe2-6, Δe2-7, Δe2-8). In contrast, the PAX5 Δe6-8, which retains the paired DNA binding domain but lacks a significant portion of the Transcriptional regulatory domain, had normal DNA binding activity. Importantly, the paired domain point mutants impaired DNA-binding in a promoter specific manner, with P80R having a marked reduction in binding to both the CD19 and mb-1 promoters, whereas P34Q showed reduced binding only to the mb-1 promoter. Surprisingly, the PAX5-ETV6 and the PAX5-FOXP1 translocations had markedly reduced DNA-binding activity despite retention of the PAX5 paired domain. As expected each of the mutants with impaired or absent DNA-binding activity were found to have markedly reduced Transcriptional activity when compared to wild type PAX5. Similarly, those mutants with altered or deleted Transcriptional activation domains had reduced Transcriptional activity, as did the two PAX5 translocation-encoded fusion proteins ( PAX5-ETV6 and PAX5-FOXP1 ). Moreover, transfection of increasing amounts of PAX5-ETV6 or PAX5-FOXP1 together with a fixed amount of wild type PAX5 revealed that the fusion proteins competitively inhibit the Transcriptional activation of wild type PAX5. Taken together, these data indicate that the identified PAX5 mutations impair DNA-binding and/or Transcriptional activity. This loss of normal PAX5 function in turn would contribute to the observed arrest in B-cell development seen in ALL.

James Hagman - One of the best experts on this subject based on the ideXlab platform.

  • hands on regulation of b cell development by the Transcription Factor PAX5
    Immunity, 2007
    Co-Authors: James Hagman, Kara Lukin
    Abstract:

    The Transcription Factor PAX5 is a critical regulator of B lymphocyte commitment and identity. In this issue of Immunity, Schebesta et al. (2007) demonstrate how PAX5 activates 170 different genes involved in B cell signaling, adhesion, migration, and maturation.

  • Transcription Factor PAX5 bsap transactivates the rag mediated v h to dj h rearrangement of immunoglobulin genes
    Nature Immunology, 2006
    Co-Authors: Zhixin Zhang, James Hagman, Celia R Espinoza, Zhihong Yu, Robert P Stephan, Ti He, Stuart G Williams, Peter D Burrows, Ann J Feeney, Max D Cooper
    Abstract:

    Immunoglobulin rearrangement from variable heavy chain (VH) to diversity (D)–joining heavy chain (JH), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage–specific Transcription Factor PAX5. Conversely, ectopic PAX5 expression in thymocytes promotes the rearrangement of DH-proximal VH7183 genes. In exploring the mechanism for PAX5 regulation of VH-to-DJH recombination, we have identified multiple PAX5 binding sites in the coding regions of human and mouse VH gene segments. PAX5 bound to those sites in vitro and occupied VH genes in early human and mouse B lineage cells. Moreover, PAX5 interacted with the recombination-activating gene 1 (RAG1)–RAG2 complex to enhance RAG-mediated VH recombination signal sequence cleavage and recombination of a VH gene substrate. These findings indicate a direct activating function for PAX5 in RAG-mediated immunoglobulin VH-to-DJH recombination.

Li Song Shen - One of the best experts on this subject based on the ideXlab platform.

  • expression of the Transcription Factor PAX5 in childhood acute leukemic cells
    Journal of Experimental Hematology, 2006
    Co-Authors: Bei Zhang, Qi Dong Ye, Long Jun Gu, Jing Yan Tang, Xiang Liang Yuan, Li Song Shen
    Abstract:

    : To investigate Transcription Factor PAX5 expression characteristics in childhood acute leukemic cells, expression levels of PAX5 and CD19 mRNA in 6 hematological tumor cell lines and bone marrow cells of 6 normal children, 58 de novo patients and 4 relapse acute leukemic children, including 39 cases of B-ALL, 10 cases of T-ALL and 13 cases of AML, were detected by a real-time RT-PCR. The results showed that PAX5 and CD19 mRNA expression levels were 2.35% and 2.52% in Namalwa (B-cell lines) respectively, but almost not detectable in other T- and myeloid cell lines. Among clinical samples, expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML (P = 0.029 and P = 0.013 respectively). PAX5 expression was significantly lower in T-ALL and AML than that in normal controls. The difference of PAX5 mRNA expression levels between T-ALL and AML was not significant. Individual difference of PAX5 mRNA expression levels in children with B-ALL was great. Moreover, PAX5 mRNA expressions in de novo and relapse patients with B-ALL were significantly higher than those in remission (P = 0.011 and P = 0.006 respectively). As binding sites for B-cell specific activator protein have been identified in the promoter regions of CD19, the study found that in B-ALL, there was clear correlation between the expression levels of PAX5 and CD19, which was also studied by real-time RT-PCR. It is concluded that PAX5 transcripts are readily detectable and quantifiable in clinical materials with B-ALL by real-time RT-PCR. The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.

  • expression of the Transcription Factor PAX5 bsap in childhood acute leukemia cells and haematological tumor cell lines
    Blood, 2004
    Co-Authors: Bei Zhang, Qi Dong Ye, Long Jun Gu, Jing Yan Tang, Xiang Liang Yuan, Yanxia Zhao, Li Song Shen
    Abstract:

    PAX5 gene is a paired-box PAX gene family member,and encodes the Transcription Factor BSAP(B-cell specific activator protein) which is a key regulator of B-cell development and differentiation.Dysregulation of PAX5 gene function may contribute to tumorigenesis in lymphoid malignancies.But up to now,a detailed examination of PAX5/BSAP expression in acute leukemia(mainly acute B-lineage lymphoblastic leukemia) has not been reported.In this study,a real-time RT-PCR assay for the relative quantitation of PAX5 and CD19 mRNA expression was developed.It was applied on 6 haematological tumor cell lines and bone marrow cells of 6 normal children,58 previously untreated and 4 relapse acute leukemic children,including 39 cases of B-ALL,10 cases of T-ALL,and 13 cases of AML.PAX5 and CD19 mRNA expression were detected in B-cell lines tested,but almost not in other T- and myeloid cell lines.Among clinical samples,expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML(P=0.029 and P=0.013,respectively).PAX5 expression was significantly lower in T-ALL and AML than normal controls.The mRNA levels of PAX5 between T-ALL and AML had not any difference.Individual difference of PAX5 mRNA expression levels in children with B-ALL was great.Because binding sites for BSAP have been identified in the promoters of CD19,the study found that in B-ALL,there was clear correlation between the level of PAX5 expression and that of CD19,which was also studied by real-time RT-PCR.BSAP expression by Western Blotting analysis was also performed in haematological tumor cells,including 6 haematological tumor cell lines and 4 clinical samples(2 cases of B-ALL,1 case of T-ALL,and 1 case of AML).The results of Western Blotting analysis showed a 52-KD BSAP band in B lineage cells,but not in T- and myeloid lineage cells.The intensity of BSAP bands was in accordance with PAX5 mRNA expression level detected by real-time RT-PCR.It was concluded that PAX5 transcripts are readily detectable and quantified in clinical materials with B-ALL by real-time RT-PCR.The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.

  • Expression of the Transcription Factor PAX5/BSAP in Childhood Acute Leukemia Cells and Haematological Tumor Cell Lines.
    Blood, 2004
    Co-Authors: Bei Zhang, Qi Dong Ye, Long Jun Gu, Jing Yan Tang, Xiang Liang Yuan, Yanxia Zhao, Li Song Shen
    Abstract:

    PAX5 gene is a paired-box PAX gene family member,and encodes the Transcription Factor BSAP(B-cell specific activator protein) which is a key regulator of B-cell development and differentiation.Dysregulation of PAX5 gene function may contribute to tumorigenesis in lymphoid malignancies.But up to now,a detailed examination of PAX5/BSAP expression in acute leukemia(mainly acute B-lineage lymphoblastic leukemia) has not been reported.In this study,a real-time RT-PCR assay for the relative quantitation of PAX5 and CD19 mRNA expression was developed.It was applied on 6 haematological tumor cell lines and bone marrow cells of 6 normal children,58 previously untreated and 4 relapse acute leukemic children,including 39 cases of B-ALL,10 cases of T-ALL,and 13 cases of AML.PAX5 and CD19 mRNA expression were detected in B-cell lines tested,but almost not in other T- and myeloid cell lines.Among clinical samples,expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML(P=0.029 and P=0.013,respectively).PAX5 expression was significantly lower in T-ALL and AML than normal controls.The mRNA levels of PAX5 between T-ALL and AML had not any difference.Individual difference of PAX5 mRNA expression levels in children with B-ALL was great.Because binding sites for BSAP have been identified in the promoters of CD19,the study found that in B-ALL,there was clear correlation between the level of PAX5 expression and that of CD19,which was also studied by real-time RT-PCR.BSAP expression by Western Blotting analysis was also performed in haematological tumor cells,including 6 haematological tumor cell lines and 4 clinical samples(2 cases of B-ALL,1 case of T-ALL,and 1 case of AML).The results of Western Blotting analysis showed a 52-KD BSAP band in B lineage cells,but not in T- and myeloid lineage cells.The intensity of BSAP bands was in accordance with PAX5 mRNA expression level detected by real-time RT-PCR.It was concluded that PAX5 transcripts are readily detectable and quantified in clinical materials with B-ALL by real-time RT-PCR.The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.

Sabine Strehl - One of the best experts on this subject based on the ideXlab platform.

  • the role of the janus faced Transcription Factor PAX5 jak2 in acute lymphoblastic leukemia
    Blood, 2015
    Co-Authors: Dagmar Schinnerl, Klaus Fortschegger, Maximilian Kauer, Joao R M Marchante, Reinhard Kofler, Monique Den L Boer, Sabine Strehl
    Abstract:

    PAX5-JAK2 has recently been identified as a novel recurrent fusion gene in B-cell precursor acute lymphoblastic leukemia, but the function of the encoded chimeric protein has not yet been characterized in detail. Herein we show that the PAX5-JAK2 chimera, which consists of the DNA-binding paired domain of PAX5 and the active kinase domain of JAK2, is a nuclear protein that has the ability to bind to wild-type PAX5 target loci. Moreover, our data provide compelling evidence that PAX5-JAK2 functions as a nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream signal transducers and activators of Transcription (STATs) in an apparently noncanonical mode. The chimeric protein also enables cytokine-independent growth of Ba/F3 cells and therefore possesses transforming potential. Importantly, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. Together, our data show that PAX5-JAK2 simultaneously deregulates the PAX5 downstream Transcriptional program and activates the Janus kinase-STAT signaling cascade and thus, by interfering with these two important pathways, may promote leukemogenesis.

  • PAX5 fusion genes in t(7;9)(q11.2;p13) leukemia: a case report and review of the literature
    Molecular Cytogenetics, 2014
    Co-Authors: Dagmar Denk, Jutta Bradtke, Margit König, Sabine Strehl
    Abstract:

    Background B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by recurrent genetic alterations including chromosomal translocations. The Transcription Factor PAX5, which is pivotal for B-cell commitment and maintenance, is affected by rearrangements, which lead to the expression of in-frame fusion genes in about 2.5% of the cases.

  • Abstract 2172: Functional studies on leukemic PAX5 fusion proteins
    Cancer Research, 2011
    Co-Authors: Klaus Fortschegger, Dagmar Denk, Wiebke Mensing, Sabine Strehl
    Abstract:

    Recent analyses of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have revealed that the gene encoding the Transcription Factor PAX5, which is essential for B-lymphocyte commitment and maintenance, is frequently affected by genetic alterations including mutations, deletions, and translocations. Chromosome rearrangements resulting in the expression of chimeric PAX5 fusion proteins account for about 2% of all BCP-ALL cases. In these chimeric proteins the N-terminus of PAX5 comprising the DNA-binding paired domain is always retained, but intriguingly the C-terminal fusion partners are substantially heterogeneous. A thorough characterization of PAX5 fusion proteins is, thus, required to determine their oncogenic properties and to unravel whether they affect common or distinct pathways. Analysis of the subcellular localization of tagged versions of PAX5 fusion proteins including PAX5-BRD1, PAX5-HIPK1, PAX5-POM121, and PAX5-DACH1 in HeLa cells using indirect immunofluorescence showed that all fusions were mainly nuclear. These data are in line with the presence of the DNA-binding paired domain and at least one nuclear localization signal, and provide further evidence that PAX5 fusion proteins potentially act as aberrant Transcription Factors. However, PAX5-HIPK1, which lacks the kinase domain of HIPK1, displayed a diffuse nuclear and cytoplasmic distribution pattern. Intriguingly, co-transfection of PAX5-HIPK1 and wild-type HIPK1 into HeLa cells, which normally express only low levels of HIPK1, resulted in localization of both proteins to nuclear speckles indicating that (auto-)phosphorylation by HIPK1 may target the two proteins to these PML-body-like structures. Further studies are ongoing to verify whether this is indeed the case. In addition, assessment of the self-interaction properties of the mentioned PAX5 chimeric proteins by co-immunoprecipitation (Co-IP) determined that PAX5-DACH1 was able to oligomerize. Bioinformatic analyses identified coiled-coil regions in the C-terminal Dachshund boxes of DACH1 and DACH2 as putative oligomerization motifs. Despite the differences in their self-interaction properties, chromatin-immunoprecipitation (ChIP) revealed that all examined fusion proteins were able to occupy PAX5 target loci (e.g. CD79A) suggesting that the paired domain was sufficient to recruit the proteins to the specific DNA-sequences. Together, on the one hand, our data support the current concept that all PAX5 fusion proteins share the common feature to act as dominant negative forms of PAX5. On the other hand, as well we demonstrate that the diverse fusion proteins possess distinct properties, which may be responsible for differences in the pathogenesis of the respective leukemia. This project is funded by the Austrian Science Fund FWF (P21554). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2172. doi:10.1158/1538-7445.AM2011-2172

  • Abstract 2171: The fusion protein PAX5-JAK2 constitutively activates JAK-STAT signaling
    Cancer Research, 2011
    Co-Authors: Dagmar Denk, Klaus Fortschegger, Sabine Strehl
    Abstract:

    In B-cell precursor acute lymphoblastic leukemia the Transcription Factor PAX5, a master regulator of B-cell commitment and maintenance, is recurrently fused to several different partner proteins including other Transcription Factors, structural proteins, and the tyrosine kinase (TK) JAK2. Mutations and translocations affecting JAK2 are found in a variety of hematopoietic malignancies and lead to its constitutive activation and cytokine-independent JAK-STAT signaling. The chimeric PAX5-JAK2 protein contains the DNA binding domain of PAX5 and the JH1 kinase domain of JAK2. Reciprocal JAK2-PAX5 transcripts are also expressed, and the respective JAK2-PAX5 fusion protein consists of the JAK2 JH2-JH7 domains fused to the PAX5 partial homeodomain and the transactivation and inhibitory domains. In a first step towards the understanding of the function of these chimeric proteins, their subcellular localization was determined. Transfection of tagged fusion proteins into HEK293 and HeLa cells displayed nuclear localization of PAX5-JAK2. In contrast, JAK2-PAX5 fusion proteins were mainly localized in the cytoplasm. In order to determine whether PAX5-JAK2 is tyrosine phosphorylated and whether PAX5-JAK2 or JAK2-PAX5 is capable of activating STAT proteins, intracellular phosphoprotein analyses using flow cytometry were performed. Transiently transfected unstimulated JAK2-deficient human gamma2A cells exhibited a high degree of phosphorylation of PAX5-JAK2 compared to JAK2 wild-type protein, strongly suggesting cytokine stimulation-independent TK activity of the fusion protein. Additionally, expression of PAX5-JAK2 but not JAK2-PAX5 resulted in constitutive phosphorylation of STATs 1, 3, and 5. The capability of PAX5-JAK2 to induce STAT5 was confirmed by reporter gene assays. Mutation of the respective tyrosine residues in PAX5-JAK2 completely abolished the phosphorylation of STATs demonstrating that their activation is phospho-PAX5-JAK2-dependent. As it has been shown that PAX5 fusion proteins can act as competitive inhibitors of wild-type PAX5, we also performed a CD19 luciferase assay which showed that PAX5-JAK2 has a negative effect on the activation capacity of wild-type protein. Additional analysis including gene expression profiling of patient material will be performed to further evaluate the function of the chimeric proteins. Together, PAX5-JAK2 appears to affect JAK-STAT signaling and may at the same time interfere with normal B-cell development. In contrast to ETV6-JAK2 – the only other JAK2 chimeric protein analyzed in detail to date – which resides in the cytoplasm and is tyrosine phosphorylated upon dimerization, PAX5-JAK2 localizes to the nucleus and lacks any putative self-association motif. Hence, PAX5-JAK2 represents the first nuclear JAK2-fusion protein, which constitutively activates the JAK-STAT signaling pathway. This project is funded by the Austrian Science Fund FWF (P21554). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2011-2171