Overexpression Of Pax5 Research Articles

Functional variant rs9344 at 11q13.3 regulates CCND1 expression in multiple myeloma with t(11;14).

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by cytogenetic abnormalities, such as t(11;14)(q13;q32), resulting in CCND1 overexpression. The rs9344 G allele within CCND1 is the most significant susceptibility allele for t(11;14). Sequencing data from 2 independent cohorts, CoMMpass (n = 698) and Mayo Clinic (n = 661), confirm the positive association between the G allele and t(11;14). Among 80% of individuals heterozygous for rs9344 with t(11;14), the t(11;14) event occurs on the G allele, demonstrating a biological preference for the G allele in t(11;14). Within t(11;14), the G allele is associated with higher CCND1 expression and elevated H3K27ac and H3K4me3. CRISPR/Cas9 mediated A to G conversion resulted in increased H3K27ac over CCND1 and elevated CCND1 expression. ENCODE ChIP-seq data supported a PAX5 binding site within the enhancer region covering rs9344, showing preferential binding to the G allele. Overexpression of PAX5 resulted in increased CCND1 expression. These results support the importance of rs9344 G enhancer in increasing CCND1 expression in MM.

Pax5 mediates the transcriptional activation of the CD81 gene

CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between − 130 and − 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between − 130 and − 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.

Tungsten Blocks Murine B Lymphocyte Differentiation and Proliferation Through Downregulation of IL-7 Receptor/Pax5 Signaling.

Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.

Abstract 589: DNA demethylating agent and histone deacetylation inhibitor synergistically induce paired box 5 gene expression in non-small cell lung cancer cell lines

Abstract Epigenetic abnormalities play an important role in carcinogenesis and tumor suppressor paired box 5 gene (Pax5) is one of the most frequently methylated genes in non-small cell lung cancer (NSCLC). Depletion of Pax5 increased malignant characteristics including proliferation, migration and invasion in mammary cancer cells, while overexpression of Pax5 does not lead to cancer in vivo and induces apoptosis in hematological cancer cells. In this study, we found that Pax5 expression was frequently absent or down-regulated in NSCLC cell lines in vitro. We tested whether a combination of DNA demethylating agent Azacytidine and histone deacetylation (HDAC) inhibitor Vorinostat has a synergistic effect on the Pax 5 gene expression and cell growth inhibition of human NSCLC cell lines. We found that the combination induced Pax5 expression synergistically in NSCLC cell lines. Azacytidine decreased global DNA methylation and reduced CpG sites methylation rate in Pax5 promoter region. Vorinostat inhibited HDAC activity, but did not directly enhance the demethylation function of Azacytidine. Further investigation is needed to uncover the mechanism of action of the synergistic effect of these two agents on the Pax5 gene expression. Our results also show that different NSCLC cell lines respond similarly to the combination treatment under our experimental conditions. Citation Format: Yuanxin Liang, Roman Perez-Soler, Yiyu Zou. DNA demethylating agent and histone deacetylation inhibitor synergistically induce paired box 5 gene expression in non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 589. doi:10.1158/1538-7445.AM2014-589

PAX5 overexpression is not enough to reestablish the mature B-cell phenotype in classical Hodgkin lymphoma

PAX5 overexpression is not enough to reestablish the mature B-cell phenotype in classical Hodgkin lymphoma

Prevention of Bone Loss after Ovariectomy in Mice with Preferential Overexpression of the Transcription Factor Paired Box-5 in Osteoblasts

We have recently shown that the transcription factor Paired box-5 (Pax5) promotes bone formation in vivo and osteoblastogenesis in vitro. Here, we demonstrated the involvement of Pax5 in bone remodeling after ovariectomy (OVX). A significant increase was seen in vertebrae bone volume in transgenic mice preferentially overexpressing Pax5 in osteoblasts by using the mouse α1(I)Collagen promoter, whereas OVX significantly reduced vertebrae bone volume in wild-type (WT) mice without significantly affecting that in Pax5 transgenic mice. Preferential osteoblastic Pax5 overexpression invariably led to significant increases in osteoblastic and osteoclastic parameters in mice with sham operation. However, OVX significantly increased osteoclastic parameters in WT mice, without additionally increasing osteoblastic and osteoclastic parameters in Pax5 transgenic mice. These results suggest that osteoblastic Pax5 would play a role in OVX-induced bone loss through a mechanism relevant to the promotion of both osteoblastic bone formation and osteoclastic bone resorption in vivo.

The transcription factor paired box-5 promotes osteoblastogenesis through direct induction of Osterix and Osteocalcin

Although skeletal abnormalities are seen in mice deficient of particular paired box (Pax) family proteins, little attention has been paid to their role in osteoblastogenesis so far. Here, we investigated the possible involvement of several Pax family members in mechanisms underlying the regulation of differentiation and maturation of osteoblasts. Among different Pax family members tested, Pax5 was not markedly expressed in murine calvarial osteoblasts before culture, but progressively expressed by osteoblasts under differentiation toward maturation. Immunoreactive Pax5 was highly detectable in primary cultured mature osteoblasts on immunoblotting and in osteoblastic cells attached to cancellous bone in mouse tibial sections on immunohistochemistry, respectively. Knockdown by small interfering RNA (siRNA) of endogenous Pax5 led to significant inhibition of the expression of Osteocalcin, and Osterix through deterioration of gene transactivation, in addition to a1(I)Collagen expression and alkaline phosphatase (ALP) staining, without affecting runt-related transcription factor-2 (Runx2) expression and cell viability in osteoblastic MC3T3-E1 cells. The introduction of Pax5 enhanced promoter activities of Osteocalcin and Osterix in a manner dependent on the paired domain in MC3T3-E1 cells. Putative Pax5 binding sites were identified in the 5'-flanking regions of mouse Osteocalcin and Osterix, whereas chromatin immunoprecipitation assay revealed the direct binding of Pax5 to particular regions of Osteocalcin and Osterix promoters in MC3T3-E1 cells. Overexpression of Pax5 significantly increased Osteocalcin, Osterix, and a1(I)Collagen expression, ALP activity, and Ca(2+) accumulation, without affecting Runx2 expression, in MC3T3-E1 cells. In vertebrae of transgenic mice predominantly expressing Pax5 in osteoblasts, a significant increase was seen in the ratio of bone volume over tissue volume and the bone formation rate. These findings suggest that Pax5 could positively regulate osteoblastic differentiation toward maturation in vitro, in addition to promoting bone formation and remodeling in vivo, as one of the transcription factors essential for controlling osteoblastogenesis independently of Runx2.

The role of Pax5 and E47 on AID expression in B lymphocytes of Aged BALB/c Mice (61.1)

Abstract Previous results from our laboratory demonstrated that stimulated enriched (95%) B cells from senescent mice (19 to 24 months) have an intrinsic defect in class switch recombination (CSR), and a decrease seen in both Pax5 and E47. The decrease in both Pax5 and E47 is proportional to the decrease in activation induced cytidine deaminase (AID) with aging. Binding sites for both Pax5 and E47 have been identified in the gene for AID, Aicda, and stimulation of B cells results in the recruitment of Pax5 and E47 to the Aicda gene and its expression. Our current results show that overexpression of both E47 and Pax5 rescues levels of AID and CSR in old B lymphocytes to young-like levels. Furthermore, exogenous retroviral E47 mRNA is more stable than the endogenous E47 due to the removal of the 3’UTR. Overexpression of Pax5 leads to up-regulation of E47 in both primary B cells and 293T cells; however, overexpression of E47 up-regulates Pax5 in B cells but not in 293T cells. Nuclear extracts from B cells of senescent mice show lower levels of binding consistent with reduced levels of E47 and Pax5 in aging. Using a DsRed lentiviral vector driven by the Aicda promoter and containing the E47 and Pax5 enhancer region, we show that in 293T cells DsRed is only expressed when the cells are co-transduced with E47 and/or Pax5.

The Pax5 Fusion Product Pax5-C20orf112 Causes Downregulation of Pre-B Cell Receptor Genes and Induces Differential Proliferation Patterns in B-Lymphoblastic Cell Lines.

Abstract 1284Poster Board I-306Recent SNP array analyses of B-acute lymphoblastic leukemia (B-ALL) have identified disruptions of the gene encoding the B-cell specific transcription factor Pax5 as one of the most common genomic lesions in this disease (> 30%); it being hemizygously deleted, mutated or involved in translocations. Pax5 translocates and forms fusion products with at least 12 different partners including C20orf112, leading to a chimeric Pax5/C20orf112 (Pax5/C20s) protein. Pax5 fusion products act as dominant negatives, competing for promoter binding sites with wild type (wt) Pax5 and thereby deregulating expression of target genes. In order to elucidate the molecular effects of fusion products involving the Pax5 gene, we performed a global gene expression analysis in the Nalm-6 B-ALL cell line. The cells were transfected with MSCV expression plasmids containing either empty vector, wild type Pax5 or a short fusion product of Pax5 and C20orf112 (Pax5/C20s), each containing IRES sequences for co-expression of GFP. Overexpression of Pax5 and Pax5/C20s was confirmed by western blot and quantitative RT PCR. RNA was extracted from cells sorted by FACS for GFP and processed for hybridization on Affymetrix HG-U133 plus 2 gene expression microarrays. Candidate genes were validated with RT real time PCR. Among the most differentially downregulated genes by the Pax5/C20s fusion product were candidate genes such as pleckstrin homology domain containing, family A member 2 (PLEKHA2) (12.64-fold), B-cell associated transcription factors POU class 2 associating factor 1 (POU2AF1) (4.4-fold) and transcription factor 3 (TCF3, E2A) (3.9-fold). Another intriguing observation was the downregulation of a group of genes associated with signaling through the pre-B cell receptor such as phosphoinositide-3-kinase adaptor protein 1 (BCAP) (3.35 fold), immunoglobulin heavy locus (IGH) (2.8 fold), pre-B lymphocyte genes -3 and -1 (VPREB3, VPREB1) (2.6-fold and 1.75-fold, respectively), spleen tyrosine kinase (SYK) (1.6 fold) and B-cell linker (SLP65, BLNK) (1.5-fold) by the Pax5/C20s fusion product. For stable expression and growth curves, Nalm6, 697, Kasumi2, RCH-ACV, SEM, HPB-Null, BV173 and BEL1 B-lymphoblastic cell lines were infected with retroviruses expressing the above mentioned retroviral expression constructs. We noted that forced expression of the PAX5/C20s fusion product inhibited growth in cell lines, which had functional pre-B cell receptor signaling. In contrast, the fusion gene either did not affect or enhanced growth of B-ALL cell lines, in which expression of a functional pre-B cell receptor was missing. Of note, Pax5 wt caused growth inhibition in B-ALL cell lines lacking functional pre-B cell receptor signaling. In cells with functional pre-B cell signaling, the response to engagement of the receptor as measured by calcium flux assay was diminished by overexpression of the Pax5/C20s fusion product as compared to empty vector control or PAX5 wt. These results suggest that the mechanisms of leukemogenesis of Pax5/C20s in ALL cells may be dependent on the functionality of the pre-B cell receptor pathway. This could be of great therapeutic value as it would potentially allow ALL cells to be divided into two different subtypes depending on pre-B cell receptor functionality and possibly identify the pre-B cell receptor pathway as a new therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6

PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.

Pax5 acts as a negative regulator for p53 (84.2)

Abstract Pax5/BSAP is an important regulator for B lineage cell development and function. Pax5 activates many B lineage specific genes expression, suppresses lineage non-appropriate genes expression, and controls B lineage specific immunoglobulin gene VH→DJH recombination. On the other hand, abnormal Pax5 expression has been found in different type of tumors, suggesting that Pax5 might have oncogenic potential. It has been previously shown that Pax5 interferes with p53 function through inhibition p53 gene transcription. Here, we show that Pax5-inhibits p53-meidated induction of different p53-responsive reporter genes without affecting the p53 protein expression level; conversely, forced expression of p53 or activation of p53 by DNA damaging response also suppresses Pax5-mediated transcriptional activation. Co-immunoprecipitation studies provide the evidence that Pax5 interacts with p53, which is essential for the mutual functional interference between these two factors. Moreover, overexpression of Pax5 attenuates DNA damage induced p53 Ser-18 phosphorylation and inhibits p53-medaited induction of downstream target genes, including p21, Bax, Puma, and Gadd45α. Through interfering p53 function, overexpression of Pax5 protects cells from DNA damage induced apoptosis. These results reveal that Pax5 acts as a negative regulator for p53, which might be essential for the dual roles of Pax5 in B lymphopoiesis and tumorigenesis.

The expression of PAX5, p53 immunohistochemistry and p53 mutation analysis in superficial bladder carcinoma tissue. Correlation with pathological findings and clinical outcome.

The expression pattern of PAX5 in the tissue of superficial bladder transitional cell carcinoma (TCC), its prognostic value and its correlation with p53 immunohistochemistry and p53 mutation analysis were evaluated. Study comprised 61 patients with histologically confirmed superficial bladder TCC. Expression level of PAX5 mRNA was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and determined semiquantitatively. The presence of p53 mutations was determined by SSCP and confirmed by direct sequencing. The p53 immunohistochemistry was performed with DO1 antibody and semiquantitatively evaluated using HSCORE (HS) method. As the control group for the evaluation of the PAX5 expression served 8 men with benign prostatic hyperplasia. PAX5 expression was found in 50 patients with bladder TCC but in no patient from the control group. Its quantity however correlated neither with the stage nor with the grade of the tumor. P53 mutation was confirmed only in 1 patient with pTaG2 tumor in exon 5 (deletion of proline 128). On the contrary, positive immunohistochemical staining of p53 was detected in most patients. Using the cutoff value of HS 200, 56.9% of patients showed p53 overexpression. Quantity of p53 immunochistochemical positivity did not correlate with the quantity of PAX5 expression. Using the cutoff values of HS 200 for p53 and of 0.2 for PAX5, 7 of 8 patients with future progression had p53 and 4 had PAX5 overexpression respectively. The expression of gene PAX5 is a frequent event in superficial TCC of the bladder.

Overexpression of Pax5 is not sufficient for neoplastic transformation of mouse neuroectoderm.

The developmental control genes of the Pax family are essential for brain development. Several Pax genes are also involved in chromosomal translocations causing malignancies in humans, and Pax5 expression is deregulated in medulloblastomas. We have investigated whether Pax5 can induce tumors in the developing mouse brain. Primary mouse embryonic neuroectodermal cells were retrovirally transduced with mouse Pax5 and transplanted into the brain of syngeneic host mice. No tumors developed in 36 transplants after one year, and there were no alterations in the differentiation pattern of the neural transplants. We then generated transgenic mice expressing human Pax5 under control of the Engrailed-2 promoter, which is expressed in the cerebellar external granule cell layer and in medulloblastomas. Sustained expression was achieved in the cerebellum of transgenic animals throughout lifetime. Expression levels were similar to those observed in human medulloblastomas. Again, cerebellar morphogenesis was undisturbed, and no tumors arose. These results strongly argue against a dominant transforming activity of PAX5 in NEC and in cerebellar granule cell precursors of mice, and underline the restricted tissue-specificity of PAX5 related oncogenesis.

Deregulated PAX-5 Transcription From a TranslocatedIgH Promoter in Marginal Zone Lymphoma

The PAX-5 gene codes for the transcription factor BSAP, which is expressed throughout B-cell development. Although loss-of-function mutation in the mouse showed an essential role forPax-5 in early B lymphopoiesis, gain-of-function mutations have implicated the human PAX-5 gene in the control of late B-cell differentiation. PAX-5 (on 9p13) has been involved together with the immunoglobulin heavy-chain (IgH) gene (on 14q32) in the recurring t(9;14)(p13;q32) translocation that is characteristic of small lymphocytic lymphoma with plasmacytoid differentiation. Here we have characterized a complex t(2;9;14)(p12;p13;q32) translocation present in a closely related non-Hodgkin’s lymphoma referred to as splenic marginal zone lymphoma (MZL). In this MZL-1 translocation, the two promoters of PAX-5 were replaced on the derivative chromosome 14 by an immunoglobulin switch Sμ promoter that was linked to the structural PAX-5 gene upstream of its translation initiation codon in exon 1B. Expression analyses confirmed thatPAX-5 transcription was upregulated due to efficient initiation at the Sμ promoter in the malignant B lymphocytes of patient MZL-1. For comparison we have analyzed PAX-5 expression in another B-cell lymphoma, KIS-1, indicating that transcription from the distalPAX-5 promoter was increased in this tumor in agreement with the previously characterized translocation of the immunoglobulin Eμ enhancer adjacent to PAX-5 exon 1A. In both lymphomas, the J-chain gene, which is thought to be under negative control by BSAP, was not expressed, whereas transcription of the putative target genep53 was unaffected by PAX-5 overexpression. Together these data indicate that the t(9;14)(p13;q32) translocation contributes to lymphoma formation as a regulatory mutation that leads to increasedPAX-5 expression in late B-cell differentiation due to promoter replacement or enhancer insertion.

Deregulated PAX-5 Transcription From a TranslocatedIgH Promoter in Marginal Zone Lymphoma

The PAX-5 gene codes for the transcription factor BSAP, which is expressed throughout B-cell development. Although loss-of-function mutation in the mouse showed an essential role for Pax-5 in early B lymphopoiesis, gain-of-function mutations have implicated the human PAX-5 gene in the control of late B-cell differentiation. PAX-5 (on 9p13) has been involved together with the immunoglobulin heavy-chain (IgH) gene (on 14q32) in the recurring t(9;14)(p13;q32) translocation that is characteristic of small lymphocytic lymphoma with plasmacytoid differentiation. Here we have characterized a complex t(2;9;14)(p12;p13;q32) translocation present in a closely related non-Hodgkin's lymphoma referred to as splenic marginal zone lymphoma (MZL). In this MZL-1 translocation, the two promoters of PAX-5 were replaced on the derivative chromosome 14 by an immunoglobulin switch Smicro promoter that was linked to the structural PAX-5 gene upstream of its translation initiation codon in exon 1B. Expression analyses confirmed that PAX-5 transcription was upregulated due to efficient initiation at the Smicro promoter in the malignant B lymphocytes of patient MZL-1. For comparison we have analyzed PAX-5 expression in another B-cell lymphoma, KIS-1, indicating that transcription from the distal PAX-5 promoter was increased in this tumor in agreement with the previously characterized translocation of the immunoglobulin Emicro; enhancer adjacent to PAX-5 exon 1A. In both lymphomas, the J-chain gene, which is thought to be under negative control by BSAP, was not expressed, whereas transcription of the putative target gene p53 was unaffected by PAX-5 overexpression. Together these data indicate that the t(9;14)(p13;q32) translocation contributes to lymphoma formation as a regulatory mutation that leads to increased PAX-5 expression in late B-cell differentiation due to promoter replacement or enhancer insertion.