Pax5 Isoforms Research Articles

Paired box gene 5 isoforms A and B have different functions in transcriptional regulation of B cell development-related genes in immature B cells.

The transcription factor paired box gene 5 (Pax5) is essential for B cell development. In this study, complementation analyses in Pax5-deficient DT40 cells showed that three Pax5 isoforms Pax5A, Pax5B and Pax5BΔEx8 (another spliced isoform of Pax5B lacking exon 8) exhibit distinct roles in transcriptional regulation of six B cell development-related genes (activation-induced cytidine deaminase, Aiolos, BTB and CNC homology 2, B cell lymphoma-6, early B cell factor 1, origin binding factor-1 genes), transcriptions of which are remarkably down-regulated by Pax5-deficiency. Moreover, ectopic expression study shows that these Pax5 isoforms may regulate themselves and each other at the transcriptional level.

Spleen-specific isoforms of Pax5 and Ataxin-7 as potential proteomic markers of lymphoma-affected spleen.

The splenomegaly, enlargement of spleen, has been observed in several diseases. It has been intended to evaluate histochemical alterations, spleen-specific enzymatic and proteomic markers during splenomegaly, and lympho-proliferative disorders from spleen of mice bearing Dalton's lymphoma. The higher expression of c-fos, c-jun, and MAPK testifies proliferation of lymphocytes. The lower expression of Pax5, higher expression of CD3, and the presence of additional form of Zap-70 suggest hypertrophy of follicles and splenomegaly influenced by weak B-cell receptor-mediated signaling, but activated T-cell receptor-mediated signaling. Simultaneously, lower levels of SOD, NDR2, and MIB2 and higher expression levels of Ataxin-7 and LDH also suggest impact of stress either as a cause or effect of cell proliferation. Spleen-specific isoform of Pax5, NDR2, MIB2, and Ataxin-7 can be considered as spleen-specific unique molecular markers for the evaluation of splenomegaly and lympho-proliferative disorders.

Alternative splicing of the trout Pax5 gene and identification of novel B cell populations using Pax5 signatures

Pax5 is an alternatively spliced transcription factor that regulates B cell development and activation. The function of specific Pax5 isoforms is unknown. Here we report the existence of seven alternatively spliced isoforms of Pax5 in the rainbow trout. We hypothesized that B cells differentially express specific Pax5 isoforms as a means of modulating Pax5 activity during cell maturation. Flow cytometric analyses using Pax5-specific antibodies recognizing the paired domain, a central (exon 6-encoding) domain, or the C-terminus, revealed the existence of distinct Pax5-expressing cell populations in trout immune tissues. Additionally, using the transcription factor EBF, we show that Pax5 isoforms lacking a paired domain are already expressed at the earliest stages of trout (B) lymphopoiesis, and unexpectedly, that minor populations of such cells reside in blood and spleen. These data support use of differentially expressed Pax5 isoforms to identify novel B cell subsets in the form of Pax5 tissue signatures, and as such, provides new biomarkers for malignancy, infectious disease, and disease resistance in trout and humans.

Isoforms of Pax5 and co-regulation of T- and B-cells associated genes influence phenotypic traits of ascetic cells causing Dalton's lymphoma

The Pax5 and its isoforms influence proliferation of B- and T-cells, during development and oncogenesis but molecular mechanism and host–tumor relationship is not clear. This report describes status of Pax5 isoforms and co-regulation of molecular markers of ascite cells causing Dalton's lymphoma in murine. Higher expressions of Pax5, CD19, CD3, Ras and Raf were observed in DLA cells. The levels of transcripts as well as p53 protein were also higher in DLA cells. The transcript of p53 from DLA cells was a variant of p53 having deletion of 50bp as compared to control. On annotation, it reflects transformation related protein p53 pseudogene mRNA. Lower level of superoxide dismutase (SOD) indicates oxidative stress and higher level of LDH5 in DLA cells reflects hypoxia in cancerous condition. The expression of Pax5d/e isoforms in DLA cells suggests presence of resting B-cells. Thus, isoforms of Pax5 and co-regulation of T- and B-cells associated genes influence phenotypic traits of ascetic cells causing Dalton's lymphoma.

Paired box gene 5 may modulate Proviral Integration of Moloney virus2gene and protein expression in mature B-cells

Deregulated transcription factor Paired box gene 5 (Pax-5) expression has been implicated in B-cell lymphoma in human disease. Proviral Integration of Moloney virus 2 (Pim-2) kinase may play a role in apoptosis and chemotherapy resistance. We characterized the expression of Pax-5 and Pim-2 in mature B-cell lines and 293T cells transfected with human isoforms of Pax-5. By quantitative real-time polymerase chain reaction (qRT-PCR), we showed that Pax-5 isoforms significantly increased Pim-2 mRNA expression in 293T cells and that mRNAs for both Pax-5 and Pim-2 were two-fold significantly expressed in mature malignant B-cells. Western blot analysis showed that Pim-2 protein expression was increased in 293T cells transiently transfected with human isoforms of Pax-5 and that Pax-5 isoforms and Pim-2 proteins were expressed in mature B-cell lines. By electrophoretic mobility shift analysis, we showed that Pax-5 isoforms were able to bind to the Pim-2 promoter region. Taken together, these results suggest that Pax-5 may act as a transcription factor to modulate the expression of Pim-2 and that both proteins may play a role in the oncogenesis of mature B-cell lymphoma.

Acute Lymphoblastic Leukemia-Associated PAX5 Mutations Induce Aberrant B Cell Development

AbstractAbstract 10Over 60% of pediatric B progenitor acute lymphoblastic leukemia (ALL) cases contain somatic mutations in genes regulating B cell development, with PAX5 being the most common target of mutation (>32% of cases). The transcription factor PAX5 is required for commitment and maintenance of the B lymphoid lineage. A variety of PAX5 mutations has been identified including, mono-allelic deletions, sequence mutations, internal deletions, frame-shift mutations and translocations. We have previously shown that these PAX5 mutations result in reduced transcriptional activity either as a result of haploinsufficiency or the generation of altered PAX5 isoforms with reduced DNA-binding and/or transcriptional activity. However, the direct effect of the mutations on normal B cell development remains unknown. To address this question, we assessed the ability of a series of PAX5 mutations to rescue normal B cell development in Pax5-/- bone marrow (BM) cells using a murine in vitro culture system.Whole BM or transduced cells were grown in IL-7 producing stromal-supported cultures for two weeks and then assessed for their extent of B cell differentiation using flow cytometry. Under these in vitro conditions, both Pax5+/+ and Pax5+/− BM cells differentiated to a Hardy fraction D pre-B cell stage of differentiation (CD43−/B220+/CD19+/BP1+), with only a slight decrease in the level of expression of BP1 detected in the Pax5+/− cells. By contrast, Pax5-/- cells failed to undergo significant differentiation under these in vitro growth conditions and were arrested at an early pro-B stage of development (CD43+/−/B220+/CD19−/BP1−). To assess the biological activity of the identified PAX5 mutants, we then transduced lineage-depleted BM cells from Pax5+/+, Pax5+/− and Pax5-/- mice with MSCV-based retroviral vectors expressing either wild type (WT) or mutant PAX5 followed by in vitro culture. Three classes of PAX5 mutations were assessed: DNA binding domain mutations (P80R, P34Q, and V26G), an internal deletion mutation (Δe6-8), and translocation-induced PAX5 chimeric genes (PAX5-ETV6, PAX5-FOXP1 and PAX5-ZNF521). As expected, expression of WTPAX5 resulted in full rescue of Pax5-/- cells and induced no significant effects on the ability of Pax5+/+ and +/− cells to differentiate. By contrast, PAX5 DNA-binding domain mutants resulted in only partial rescue of Pax5-/- cells, with P80R inducing B220+/CD19−/BP1−, P34Q producing B220+/CD19+ cells with weak BP1 expression, and V26G yielding CD19+/BP1+ cells with minimally reduced levels of BP1. Similarly, expression of Δe6-8 resulted in partial rescue with the expansion of B220+/CD19+/−/BP1− cells. In stark contrast, expression of the translocation encoded PAX5 fusion proteins failed to induce any evidence of rescue. Moreover, these fusion proteins induced only minimal perturbations in the ability of Pax5+/+ and +/− cells to differentiate, suggesting that these fusion proteins were weak competitive inhibitors of normal Pax5 transcriptional activity under intra-cellular conditions. To further characterize the effects of these PAX5 mutations on B cell differentiation, we next analyzed the gene expression patterns of the resultant cell populations using the Mouse Genome 430 2.0 Arrays (Affymetrix) and compared the profiles to those obtained from purified Hardy fractions from normal murine BM. The expression signatures of the Pax5-/- cells were identical to those for normal Hardy fraction A and shifted to the signature of Hardy fraction C following rescue with WTPAX5. Transduction of Pax5-/- cells with either V26G or P34Q resulted in a near complete rescue with expression signatures similar to those obtained for Hardy fractions B/C. By contrast, transduction with P80R or Δe6-8 yielded a more incomplete rescue with expression profiles that were between Hardy fractions A and B. Interestingly, a number of genes within the B cell receptor signaling pathway were altered in cells rescued by P80R and Δe6-8, including the down regulation of CD19, Btk and Blnk. In summary, our data demonstrate that leukemia-associated PAX5 mutations have a graded effect on the transcriptional network that controls normal B cell development and differentiation. Defining the differential target gene specificity of the various PAX5 mutants should provide valuable insights into the molecular mechanisms through which these genetic lesions contribute to leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

The PAX5 gene is frequently rearranged in BCR-ABL1-positive acute lymphoblastic leukemia but is not associated with outcome. A report on behalf of the GIMEMA Acute Leukemia Working Party

Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric patients with acute lymphoblastic leukemia. So far the occurrence of PAX5 alterations and their clinical correlation have not been investigated in adults with BCR-ABL1-positive acute lymphoblastic leukemia. The aim of this study was to characterize the rearrangements on 9p involving PAX5 and their clinical significance in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Eighty-nine adults with de novo BCR-ABL1-positive acute lymphoblastic leukemia were enrolled into institutional (n=15) or GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) (n=74) clinical trials and, after obtaining informed consent, their genome was analyzed by single nucleotide polymorphism arrays (Affymetrix 250K NspI and SNP 6.0), genomic polymerase chain reaction analysis and re-sequencing. PAX5 genomic deletions were identified in 29 patients (33%) with the extent of deletions ranging from a complete loss of chromosome 9 to the loss of a subset of exons. In contrast to BCR-ABL1-negative acute lymphoblastic leukemia, no point mutations were found, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1-positive acute lymphoblastic leukemia. The deletions were predicted to result in PAX5 haploinsufficiency or expression of PAX5 isoforms with impaired DNA-binding. Deletions of PAX5 were not significantly correlated with overall survival, disease-free survival or cumulative incidence of relapse, suggesting that PAX5 deletions are not associated with outcome. PAX5 deletions are frequent in adult BCR-ABL1-positive acute lymphoblastic leukemia and are not associated with a poor outcome.

Highlights of This Issue

Small molecules targeting Aurora A kinase have entered Phase I/II clinical trials. Here we show for the first time that inhibition of Aurora A kinase, using the small molecule inhibitor MLN8054, results in senescence both in vitro and in vivo. This example of chemotherapy-induced senescence adds to a growing list of chemotherapies shown to induce senescence in preclinical models. Senescence is just beginning to be understood as a common chemotherapy-induced cancer cell fate. With this awareness, it is likely that senescence will be used more frequently as a biomarker in the clinic.Myc suppression of the cell cycle inhibitor p27Kip1 involves the E3 ubiquitin ligase SCFSkp2. In the absence of either Skp2 or the small adaptor protein Cks1, SCFSkp2 is inactivated and p27Kip1 accumulates despite the presence of oncogenic Myc levels. In vivo, Skp2 deficiency has surprisingly modest effects on tumorigenesis in the Eμ-Myc lymphoma model, in contrast to previously shown delayed tumor onset in Cks1-deficient mice. These data genetically support clinical observation that show that high Skp2 levels are not always associated with decreased p27Kip1 expression. The results strongly support SCFSkp2-independent functions of Cks1 in tumor pathogenesis.Both β-catenin and BRCA1 proteins are necessary for normal mammary gland development, whereas their aberrant activation was shown to be involved in carcinogenesis. We show a novel association between BRCA1 and β-catenin and that BRCA1 regulates the active form of β-catenin during oxidative stress responses. This form of β-catenin in the nucleus was absent or expressed at low levels in most BRCA1 familial breast tumors, compared with its expression in sporadic breast tumors and normal breast tissue. Thus, loss of BRCA1 leads to impaired expression of the nuclear form of β-catenin, which may contribute to the pathogenesis of breast cancer.Increased PAX5 expression has been reported for a variety of human malignancies, including metastatic breast cancer. However, the functional role of PAX5 in cancer remains unclear with conflicting reports of both tumor promoting and tumour suppressor functions. Vidal and colleagues investigated the role of one PAX5 isoform, PAX5α in breast cancer cell lines and observed that PAX5α significantly reduced cell oncogenicity, tumor growth, and enhanced epithelial cell characteristics. Thus, PAX5α promotes mesenchymal to epithelial transition, a process implicated in the establishment of distant metastases.

Multiple isoforms of PAX5 are expressed in both lymphomas and normal B‐cells

The transcription factor Pax5 plays a critical role in B cell development. It has been shown that alternative splicing of its gene (PAX5) produces several distinct transcripts that modify the amino acid sequence of the putative Pax5 proteins. Subsequent studies have attempted to correlate the expression of PAX5 isoforms with certain B-cell lymphomas, the conclusions of which suggest that altered isoform expression is involved in lymphomagenesis. However, in the absence of definitive data for PAX5 isoform expression patterns in normal B cells it is difficult to confirm whether aberrant isoform expression can indeed be correlated with disease. Using a high-resolution method of analysis of reverse transcription polymerase chain reaction products, we sought to analyse the expression of the different PAX5 isoforms in normal B-cells as well as a number of B-cell lymphoma and chronic lymphocytic leukaemia cases. It was found that multiple PAX5 isoforms were expressed in both normal and malignant B cells. Immunodetection and polysomal RNA analyses also confirmed that the different PAX5 mRNAs were translated into their corresponding proteins. No consistent deregulation of PAX5 isoform expression was observed in B-cell lymphomas, but rather, complex isoform expression patterns were found in normal B cell as well as B-cell lymphoma and CLL cases.

Altered mRNA Expression of Pax-5 Is a Common Event in Acute Lymphoblastic Leukemia.

Introduction.Pax-5 gene codifies for a transcription factor central to B-cell differentiation and function, expressed during the early stage of differentiation and alternatively spliced during B-cell development. In addition to the full-length isoform (Pax-5a), isoforms arising from the inclusion or exclusion of exons 2, 7, 8, and/or 9, and lacking the DNA-binding and the transactivating domains, have been described. These isoforms and their levels of expression increase during B-cell maturation. In particular, Pax-5b isoform (deleted of exon 2), resulting in protein with partial DNA-binding domain, is related with the differentiation stage. Recently, mutations of Pax-5 gene were reported in 31.7% of 242 children (Mullighan, Nature 2007) and 30% of 119 adults (Familiades, ASH 2007 abs. 2806) with B-cell precursor acute lymphoblastic leukemia (ALL). We investigated Pax-5 gene mutations and isoforms expression in patients with ALL at the diagnosis and in selected cell populations from control subjects.Methods.Pax-5 mutations and mRNA isoforms were investigated by sequence analysis. Pax-5a and Pax-5b expression levels were evaluated by quantitative PCR in leukemic cells. Total RNA was obtained from leukemic blasts of 95 patients with B-cell precursor ALL at diagnosis, 45 adults and 50 children. These last had been selected according to the presence of adverse translocations: BCR/ABL p190 (n=10), E2A-PBX1 (n=10), TEL/AML (n=10), MLL/AF4 (n=10), or none (n=10). mRNA was obtained from purified peripheral blood CD19+ lymphocytes (6 samples), from bone marrow CD34+ hematopoietic progenitor cells (6 samples), and pro-B CD34+CD19+ cells (3 samples) from a total of 8 healthy bone marrow donors.Results. Pax-5 gene point mutations were found in 8/45 adults (18%) and 14/50 children (28%) (p=n.s.), resulting in a total of 20 different mutations. Mutations were located as follows: exon 2 (53G>C, 76delG, 101C>G, 113G>A, 197G>A), exon 3 (223A>G, 247delA, 296T>C), exon 4 (446A>T, 526G>A), exon 5 (580A>C, 601G>A), exon 6 (625C>T, 716G>A), exon 7 (836C>A), exon 8 (955G>A, 964insC, 971A>G), exon 9 (1058C>T), exon 10 (1133G>A). There was no correlation between the presence of mutations and the listed genetic subtypes. Pax-5 alternative splicing was observed in 47/95 cases (49%): 29/45 adults (64%) and 18/50 children (36%) (p=n.s.). Alternative splicing resulted in increased frequencies of the Pax-5b isoform and the deletion of exons 8 and/or 9; furthermore, a novel isoform resulting from the skipping of exon 5 was documented. On the contrary, only the fulllenght isoform was present in CD34+ progenitors and CD34+CD19+ cells from healthy donors, in which deleted isoforms were detected only at the stage of mature B-lymphocytes.Conclusion: Pax-5 mutations are frequent in childhood and adult ALL and are scattered all over the gene. We extend the current knowledge on their pathogenic role by demonstrating that alternative splicing is a common event in these patients. Imbalance between the fulllength and the Pax-5b isoforms is expected to affect the maturation of the B-cell and its propensity to apoptosis (Robichaud Nucleic Acids Res 2008), thus contributing to the process of leukemogenesis. Based on our findings, this mechanism acts broadly and is independent of the most common ALL genetic translocations.

Development of an isoform-specific gene suppression system: the study of the human Pax-5B transcriptional element

The transcription factor Pax-5, is vital during B lymphocyte differentiation and is known to contribute to the oncogenesis of certain cancers. The Pax-5 locus generates multiple yet structurally related mRNA transcripts through the specific activation of alternative promoter regions and/or alternative splicing events which poses challenges in the study of specific isoform function. In this study, we investigated the function of a major Pax-5 transcript, Pax-5B using an enhanced version of the Hepatitis Delta Virus ribozyme (HDV Rz) suppression system that is specifically designed to recognize and cleave the human Pax-5B mRNA. The activity of these ribozymes resulted in the specific suppression of the Pax-5B transcripts without altering the transcript levels of other closely related Pax-5 isoforms mRNAs both in vitro and in an intracellular setting. Following stable transfection of the ribozymes into a model B cell line (REH), we showed that Pax-5B suppression led to an increase of CD19 mRNA and cell surface protein expression. In response to this Pax-5B specific deregulation, a marked increase in apoptotic activity compared to control cell lines was observed. These results suggest that Pax-5B has distinct roles in physiological processes in cell fate events during lymphocyte development.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-Mediated Impairment of B Cell Differentiation Involves Dysregulation of Paired Box 5 (Pax5) Isoform, Pax5a

The persistent environmental contaminant and immunotoxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), markedly suppresses humoral immune responses. We recently reported impaired down-regulation of paired box 5 (Pax5), a repressor of B cell differentiation and concomitant suppression of the IgM response by TCDD in the murine CH12.LX B cell line. The objectives of the current study were to determine the impact of TCDD treatment on molecular outcomes characteristic of terminal B cell differentiation and to assess the role that Pax5 isoforms plays in the suppression of B cell differentiation by TCDD. In this study, we show that the highly abundant full-length Pax5 isoform, Pax5a, and at least two additional modestly expressed Pax5 isoforms were expressed in CH12.LX and splenic B cells. In lipopolysaccharide (LPS)-activated B cells, all of the identified Pax5 isoforms were synchronously down-regulated, and in the presence of TCDD cotreatment they were abnormally and synchronously elevated, suggesting a common mechanism of regulation. Furthermore, B cell differentiation markers X-box protein-1 and major histocompatibility complex class II showed that the levels to which Pax5 was derepressed by TCDD were sufficient to impair B cell differentiation and immunoglobulin gene expression. Confirming the involvement of Pax5, ectopic expression of Pax5a in the LPS-activated CH12.LX cells closely mimicked the suppression of the IgM response by TCDD. In summary, our results demonstrate that Pax5a has a critical role in both the TCDD-mediated impairment of B cell differentiation and the suppression of the humoral immune response.

Pax5 isoform expression and mutation status in B‐cell chronic lymphocytic leukemia

Pax5 isoform expression and mutation status in B‐cell chronic lymphocytic leukemia

Frequent Occurrence of Aberrant Pax5 Splice Variants in Philadelphia Chromosome-Positive Lymphoblastic Leukemia.

Pax5 is a key regulator of B cell commitment and is indispensable throughout the early stages of B cell differentiation. B cell development is blocked at the pro-B cell stage in Pax5-null mice. On the other hand, several investigators have reported that aberrant Pax5 transcripts as well as forced expression of Pax5 result from genetic and/or epigenetic mechanisms especially in B cell malignancies, suggesting the involvement of Pax5 abnormality in development and progression of B cell malignancies. In the present study, we examined Pax5 expression in Philadelphia chromosome (Ph)-positive lymphoid malignancies including 17 cases of Ph-positive acute lymphoblastic leukemia (Ph-ALL) and 3 cases of lymphoid blast crisis of chronic myeloid leukemia (CML-BC). By the reverse transcription-polymerase chain reaction (RT-PCR) method, we detected 4 types of Pax5 isoforms among those. In 16 samples, full-length as well as truncated forms of Pax5 were expressed. Expression of Pax5 could not be detected in 3 cases, and was only faintly found in the remaining one. These 4 cases did not express or very weakly expressed EBF, early B cell transcription factor upstream of Pax5. Sequencing analysis revealed that all 4 truncated isoforms were derived from exon skipping as follows. Type I is lacking in exon 2 and exon 3 (D2/3), type II shows deletion of exons 2–6 (D2-6), type III; D2-7, and type IV; D2-8. Two isoforms resulted in frame-shift, and two isoforms were in-frame splice variants without exons coding DNA binding domain. Transactivation potential of these two in-frame variants was examined by transfection of 293T cells with a reporter plasmid containing high-affinity Pax5 binding sites from the CD19 gene linked to the minimal TATA-like promoter, indicating much lower transcriptional activity in comparison to wild-type Pax5. These results suggest the frequent occurrence in adult Ph-positive lymphoid malignancies of aberrant Pax5 splice variants with reduced transcriptional activity, leading to impairment of normal B cell differentiation.

Identification and comparative analysis of Pax5 C-terminal isoforms expressed in human cord blood-derived B cell progenitors

We identified three Pax5 isoforms due to alternative splicing of the C-terminal exons of its gene in cord blood (CB)-derived B cell progenitors cultivated on the murine bone marrow stromal (HESS-5) cells. Apart from wild type (wt), one isoform skips exon 9 without subsequent frameshift (del9), while the other has a frameshift insert between exons 8 and 9, resulting in novel C-terminal sequences (ins8′). Quantitative reverse transcription-polymerase chain reaction analysis revealed that wt mRNA could be detected in CB CD34 + cells, but that del9 and ins8′ isoforms only appeared after 1 or 3 weeks of co-culture, respectively. Expression of each isoform mRNA was markedly upregulated during B cell differentiation in vitro, and wild type continued to be the most abundant isoform. In a luciferase reporter assay using a synthetic CD19 enhancer, del9 isoform revealed slightly lower activity and ins8′ isoform showed much lower activity, compared with Pax5-wt. Furthermore, retroviral expression of each Pax5 isoform in CB CD34 + cells induced aberrant CD19 expression in a fraction of immature myeloid cells after 1 week of culture, although del9 and ins8′ isoforms showed much less potent activity than Pax5-wt. These results suggest that Pax5-wt is quantitatively and qualitatively dominant over other C-terminal isoforms during human B cell differentiation.

Expression of mRNA for a newly identified Pax5 exon is reduced in multiple myeloma

Pax5 is a transcription factor that is critical in the bone marrow for differentiation and proliferation of B cells until the plasma cell stage. In Pax5(-/-) mice, B-cell development stalls at the pro-B-cell stage. Messenger RNA profiles of alternatively spliced isoforms of Pax5 in bone marrow frequently differ between multiple myeloma (MM) patients and healthy donors. We sought to determine if Pax5 mRNA profiles also differed in blood and unexpectedly detected the presence of a previously unreported exon that alters the amino acid code for the transactivating domain of Pax5 in CD138(-) B cells. This unique exon escapes detection by conventional analyses of RT-PCR products and may serve as a prototype for other exons, in other genes, that escape RT-PCR detection. Eight percent of tested human subjects were heterozygous for an allele with a nonsynonymous nucleotide substitution in the new exon, and one MM patient was homozygotic for this base difference. Subsequent analysis of plasma and B-cell populations from bone marrow revealed a markedly reduced mRNA expression of the new isoform in cells from MM patients when compared to cells from normal subjects.

Constitutive Expression of Pax5 in Cord Blood Progenitor Cells Rather Inhibits B Lymphopoiesis as Well as Myelopoiesis through the Exon 9-Dependent and Independent Mechanism.

Pax5 is a key regulator of B cell commitment and is indispensable throughout the early stages of B cell differentiation. Human B lymphopoiesis can be simulated by cultivating cord blood (CB) progenitor cells on the murine bone marrow stromal cell line such as HESS-5. When CB CD34+ cells are seeded onto HESS-5 cells and supplemented with SCF and G-CSF, the number of non-adherent myeloid cells initially increases, reaching a maximum during the 3rd week, and decreases rapidly thereafter. On the other hand, foci of small B-lymphoid cells beneath the stromal layer appear during the 2nd week and increase in size until the 6th week. In this culture system, we have identified two C-terminal splice variants of Pax5; one skips exon 9 without subsequent frameshift (del9), and the other has a frameshift insert between exon 8 and 9, resulting in novel C-terminal sequences (ins8′). By quantitative RT-PCR analysis, del9 mRNA was barely detected in CB CD34+ cells and thereafter upregulated in progenitor B cells during the culture. In contrast, wild-type Pax5 (wt) and ins8′ mRNA could not be detected until the 3rd week. Then, wt mRNA was rapidly upregulated 100-fold or more abundantly than del9 and ins8′ mRNA (Fig. 1). Transactivation potential of Pax5 isoforms was examined with a reporter plasmid containing high-affinity Pax5 binding sites from the CD19 gene linked to the minimal TATA-like promoter. As a result, del9 showed constantly higher (up to 3.5-fold) activity, but ins8′ had slightly lower activity than wt Pax5. Furthermore, TLE4, a Groucho co-repressor family member and a known Pax5 partner, inhibited the transcriptional activity of wt Pax5 but not del9 or ins8′ isoform, suggesting its interaction with the exon 9 sequences of Pax5. We next examined the impact of Pax5 on growth and differentiation of CB CD34+ cells by retroviral transduction of each isoform as well as EGFP. After infection, sorted EGFP + cells were subjected to the colony-forming assay and the co-culture assay on HESS-5 cells. Erythroid or mixed colony formation was almost completely inhibited by either isoform. GM-colony formation was moderately inhibited by del9 and ins8′ isoforms, but profoundly suppressed by wt Pax5. In coculture with HESS-5 cells, the number of non-adherent myeloid cells was reduced by Pax5 isoforms in a similar manner to the results of GM-colony formation. Unexpectedly, after 6 weeks of culture, the number of CD19+ adherent lymphoid cells decreased more profoundly in the culture transduced with wt Pax5 than those with del9 and ins8′ isoforms (Fig 2). These results suggest that constitutive expression of Pax5 in cord blood progenitor cells substantially inhibits not only myelopoiesis but also B lymphopoiesis through the exon 9-dependent and independent mechanism. We also raise the possibility that developmentally regulated concentration of Pax5 isoforms may be crucial for human B lymphopoiesis. [Display omitted]

Human Pax-5 C-terminal Isoforms Possess Distinct Transactivation Properties and Are Differentially Modulated in Normal and Malignant B Cells

The transcription factor Pax-5 occupies a central role in B cell differentiation and has been implicated in the development of B cell lymphoma. The transcriptional activation function of Pax-5 requires an intact N-terminal DNA-binding domain and is strongly influenced by the C-terminal transactivation domain. We report the identification and characterization of five human Pax-5 isoforms, which occur through the alternative splicing of exons that encode for the C-terminal transactivation domain. These isoforms arise from the inclusion or exclusion of exon 7, exon 8, and/or exon 9. Three of the Pax-5 isoforms generate novel protein sequences rich in proline, serine, and threonine amino acids that are the hallmarks of transactivation domains. The Pax-5 isoforms are expressed in peripheral blood mononuclear cells, cancerous and non-cancerous B cell lines, as well as in primary B cell lymphoma tissue. Electrophoretic mobility shift assays demonstrate that the isoforms possess specific DNA binding activity and recognize the PAX-5 consensus binding sites. In reporter assays using the CD19 promoter, the transactivation properties of the various isoforms were significantly influenced by the changes in the C-terminal protein sequence. Finally, we demonstrate, for the first time, that human Pax-5 isoform expression is modulated by specific signaling pathways in B lymphocytes.

Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma

Multiple myeloma (MM) is a plasma cell disorder that potentially initiates during an early stage of B-cell development. We encountered an unidentified isoform of B cell-specific activator protein (BSAP, or Pax5) in MM cells while performing differential analyses to compare mRNA expression in malignant and normal plasma cells. Pax5 is a transcription factor that plays a central role throughout B-cell development until the point of terminal differentiation. Our finding of this unique isoform prompted us to investigate Pax5 isoform usage in plasma cells and B-cell populations in other MM and healthy subjects. In contrast to normal Pax5 expression, we observed multiple isoforms of Pax5 in conjunction with low levels of expression of the full-length Pax5 in B cells from MM patients. The expressed isoforms in MM varied considerably from patient to patient, with no clear pattern. We also performed semiquantitative analyses of the mRNA expression levels of B lymphocyte-induced maturation protein (Blimp-1), because expression levels of Pax5 and Blimp-1 have been shown to be inversely correlated. We observed the expression of Blimp-1 in the B-cell populations in all 11 MM patients but in none of 11 healthy subjects. We hypothesize that premature Blimp-1 expression coupled to altered and deficient Pax5 expression causes some proliferating B cells to prematurely differentiate to plasma cells in MM.

Differential expression of alternative isoforms of Pax-5 during B-cell development

Differential expression of alternative isoforms of Pax-5 during B-cell development