B-cell Specific Transcription Factor Research Articles

Bifurcated BACH2 control coordinates mantle cell lymphoma survival and dispersal during hypoxia

AbstractBACH2, a B-cell–specific transcription factor, plays a critical role in oxidative stress–mediated drug resistance in mantle cell lymphoma (MCL); however, the biological functions of BACH2 and its regulation of B-cell malignancies in chronic hypoxic microenvironment have not been studied. Here, we found that silencing BACH2 led to not only increased tumor formation and colony formation but also increased tumor dispersal to spleen and bone marrow. Decreased BACH2 levels in patients were also correlated with bone marrow and gastrointestinal dispersal of MCL and blastoid subtypes of MCL. Unexpectedly, decreased BACH2 levels in dispersed MCL cells were due to direct transcriptional repression by hypoxia-induced factor 1α (HIF-1α) and increased heme-mediated protein degradation. In normoxic conditions, BACH2 was able to modulate HIF-1α degradation by suppressing prolyl hydroxylase 3 expression. Bifurcated BACH2 controls during hypoxia and normoxia coordinate not only MCL tumor dispersal but also drug resistance, including bortezomib resistance, via plasmacytic differentiation. Our data highlight an interactive relationship between tumor cells and local microenvironment and the mechanisms of B-cell transcription factor in the regulation of MCL dispersal.

Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia.

Recently, we identified deregulated expression of the B-cell specific transcription factor MEF2C in T-cell acute lymphoid leukemia (T-ALL). Here, we performed sequence analysis of a regulatory upstream section of MEF2C in T-ALL cell lines which, however, proved devoid of mutations. Unexpectedly, we found strong conservation between the regulatory upstream region of MEF2C (located at chromosomal band 5q14) and an intergenic stretch at 7q11 located between STAG3L4 and AUTS2, covering nearly 20 kb. While the non-coding gene STAG3L4 was inconspicuously expressed, AUTS2 was aberrantly upregulated in 6% of T-ALL patients (public dataset GSE42038) and in 3/24 T-ALL cell lines, two of which represented very immature differentiation stages. AUTS2 expression was higher in normal B-cells than in T-cells, indicating lineage-specific activity in lymphopoiesis. While excluding chromosomal aberrations, examinations of AUTS2 transcriptional regulation in T-ALL cells revealed activation by IL7-IL7R-STAT5-signalling and MEF2C. AUTS2 protein has been shown to interact with polycomb repressor complex 1 subtype 5 (PRC1.5), transforming this particular complex into an activator. Accordingly, expression profiling and functional analyses demonstrated that AUTS2 activated while PCGF5 repressed transcription of NKL homeobox gene MSX1 in T-ALL cells. Forced expression and pharmacological inhibition of EZH2 in addition to H3K27me3 analysis indicated that PRC2 repressed MSX1 as well. Taken together, we found that AUTS2 and MEF2C, despite lying on different chromosomes, share strikingly similar regulatory upstream regions and aberrant expression in T-ALL subsets. Our data implicate chromatin complexes PRC1/AUTS2 and PRC2 in a gene network in T-ALL regulating early lymphoid differentiation.

Aberrantly Expressed OTX Homeobox Genes Deregulate B-Cell Differentiation in Hodgkin Lymphoma.

In Hodgkin lymphoma (HL) we recently reported that deregulated homeobox gene MSX1 mediates repression of the B-cell specific transcription factor ZHX2. In this study we investigated regulation of MSX1 in this B-cell malignancy. Accordingly, we analyzed expression and function of OTX homeobox genes which activate MSX1 transcription during embryonal development in the neural plate border region. Our data demonstrate that OTX1 and OTX2 are aberrantly expressed in both HL patients and cell lines. Moreover, both OTX loci are targeted by genomic gains in overexpressing cell lines. Comparative expression profiling and subsequent pathway modulations in HL cell lines indicated that aberrantly enhanced FGF2-signalling activates the expression of OTX2. Downstream analyses of OTX2 demonstrated transcriptional activation of genes encoding transcription factors MSX1, FOXC1 and ZHX1. Interestingly, examination of the physiological expression profile of ZHX1 in normal hematopoietic cells revealed elevated levels in T-cells and reduced expression in B-cells, indicating a discriminatory role in lymphopoiesis. Furthermore, two OTX-negative HL cell lines overexpressed ZHX1 in correlation with genomic amplification of its locus at chromosomal band 8q24, supporting the oncogenic potential of this gene in HL. Taken together, our data demonstrate that deregulated homeobox genes MSX1 and OTX2 respectively impact transcriptional inhibition of (B-cell specific) ZHX2 and activation of (T-cell specific) ZHX1. Thus, we show how reactivation of a specific embryonal gene regulatory network promotes disturbed B-cell differentiation in HL.

Nuclear Translocation of B-Cell-Specific Transcription Factor, BACH2, Modulates ROS Mediated Cytotoxic Responses in Mantle Cell Lymphoma

BACH2, a B-cell specific transcription factor, plays a critical role in oxidative stress-mediated apoptosis. Bortezomib (VelcadeTM) is widely used to treat relapsed mantle cell lymphoma (MCL) patients despite varying clinical outcomes. As one of the potential mechanisms of action, bortezomib was reported to elicit endoplasmic reticulum (ER) stress which triggers reactive oxygen species (ROS). In the present study, we investigated the redox-sensitive intracellular mechanism that might play a critical role in bortezomib response in MCL cells. We demonstrated that in MCL cells that are sensitive to bortezomib treatments, BACH2 was translocated to the nucleus in response to bortezomib and induced apoptotic responses through the modulation of anti-oxidative and anti-apoptotic genes. On the other hand, in bortezomib resistant cells, BACH2 expression was confined in the cytoplasm and no suppression of antiapoptotic or antioxidative genes, Nrf2, Gss, CAT, HO-1 and MCL1, was detected. Importantly, levels of BACH2 were significantly higher in bortezomib sensitive MCL patient cells, indicating that BACH2 levels could be an indicator for clinical bortezomib responses. BACH2 translocation to the cytoplasm after phosphorylation was inhibited by PI3K inhibitors and combinatory regimens of bortezomib and PI3K inhibitors sensitized MCL cells to bortezomib. These data suggest that cellular distribution of BACH2 in response to ROS determines the threshold for the induction of apoptosis. Therapies that inhibit BACH2 phosphorylation could be the key for increasing bortezomib cytotoxic response in patients.

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells.

Genomewide Detection of Genes Targeted by Aberrant Somatic Hypermutation in Lymphoma,

Abstract 3474The processes of somatic hypermutation (SHM) and class switch recombination introduced by activation-induced deaminase (AID) are key steps for creating a pool of diversified antibodies in activated germinal center (GC) B cells (centroblasts; CBs) upon antigen dependent stimulation of resting naïve B cells (NBs) by T cells. Periodically, centroblasts enter the light zone of the GC where they interact with dendritic cells and T helper cells to undergo clonal selection for cells expressing high affinity antibodies. Unfortunately, AID can also accidentally introduce mutations at bystander loci, particularly within the promoter regions of a number of proto-oncogenes (BCL6, MYC, PAX5, PIM1, RhoH, S1PR2, and SOCS1) in diffuse large B cell lymphomas (DLBCL), an aggressive non-Hodgkins lymphomas. Since current methods, such as Exon Capture and RNAseq, only target mutations in coding regions, to date aberrant promoter SHMs have been studied only in a handful of genes. Given the complexity of lymphomagenesis, there may be additional genes that are targeted aberrantly by SHM at their promoters in DLBCLs.To identify genes that are targeted by SHM genomewide, we designed a novel approach to search for SHM hotspots in the genome by integrating bioinformatics tools with next generation sequencing technology. We developed a comprehensive software framework (called SHMseeqer) for discovery of single nucleotide variants (SNV) that have SHM features from ChIP-seq reads. We ran SHMseeqer on several histone H3 lysine 4 trimethylation (H3K4me3, preferentially enriched at promoters) ChIP-seq datasets obtained from two DLBCL cell lines and from normal primary NBs and CBs. We observed increased numbers of genes that harbor SHM at their promoters in DLBCL cells as compared to primary normal B cells. Moreover, in DLBCL cells, we observed promoter SHM hotspots in genes that have not been reported before, including BACH2, BTG2, EBF1, and TCL1A, etc., all of which have potential roles in B cell survival, differentiation, and malignant transformation. In addition, using BCL6 and BACH2 promoters as examples, we demonstrated that SHMs identified in these promoters greatly altered the promoter activities in a reporter assay. BACH2 is a B-cell specific transcription factor that plays an important role in gene expression regulation during B-cell activation and terminal differentiation. We observed that the SHM containing BACH2 promoter had increased transcription activity in the reporter assay as compared to widetype BACH2 promoter. We further showed that there was an elevated BACH2 level in DLBCL cells, and together with BCL6, BACH2 can block mature B-cell differentiation by repressing the expression of PRDM1.Our approach provides the first cost-efficient, genome-wide method to screen promoter mutations and SHM hotspots; an area that has not been fully explored thus far. Furthermore, by using this approach we showed that there were increased aberrant SHMs in DLBCL cells, suggesting that the process of AID-induced SHM is impaired in DLBCL cells. Moreover, the AID-induced promoter SHMs may deregulate the expression of key B-cell genes that leads to DLBCL transformation ultimately. Disclosures:No relevant conflicts of interest to declare.

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

NF-κB is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-κB-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor κB (IκB)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-κBs and phylogenetic analysis suggested that EsRelish was a member of the NF-κB family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

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.

Gene Expression Profiling of Persistent Polyclonal B-Cell Lymphocytosis Using Whole Genome Microarrays.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare B-cell disorder characterised by elevated serum levels of polyclonal IgM and persistent lymphocytosis of often bi-nucleated B-cells. The molecular basis of this disorder however remains poorly understood. We therefore used whole genome expression microarrays (Affymetrix U133 plus2.0) to compare the gene expression profile of 14 cases of PPBL with normal lymphocyte populations (6 B cell (CD19+) and 3 T cell (CD3+) samples). PPBL patients (10 female, 4 male) had a median age of 43 (range 28–63) and all were smokers with an average lymphocyte count of 5.34 x 107/ml (range 3.7–9) at time of presentation. Eight of these cases had +i(3q) and eight cases exhibited PCC, four cases displayed both chromosomal abnormalities. Normal lymphocyte populations were obtained from two pools of 12 individuals and individual donors.Microarray data were first RMA pre-processed and then expression levels of patient samples' genes compared with levels of both normal B cell and T cell samples. ANOVA (P<0.01) was used to identify differentially expressed genes. Thirty-six genes were found to be up-regulated and 157 genes were down-regulated in all patient samples by at least 2-fold.Components of the AP-1 transcription complex (FOS, FOSB and JUN) were found to be highly up-regulated in all PPBL samples with an average of 7.2, 7.9 and 31.6 fold expression respectively. The cell cycle regulatory molecule CDC27 was also found to be up-regulated in all patient samples (8.8-fold). Down-regulated genes of interest included the B-cell specific transcription factor BACH2 (3-fold), tumor-suppressor SMAD4 (3.6-fold) and apoptotic genes DRAK1 (3-fold) and CAS (5-fold). These data are currently being validated by quantitative RT-PCR.We propose that over-expression of AP-1 may have significance in PPBL as increased expression of components of AP-1 have previously been linked with lymphoproliferative disorders including Hodgkin's lymphoma and splenic marginal zone lymphoma. The underlying reason for over-expression of AP-1 in PPBL remains unknown but two characteristic features of this disease could provide clues; firstly AP-1 up-regulation has been demonstrated to be tobacco-induced in animal models and secondly the LMP1 protein of EBV can result in constitutive expression of the AP-1 complex.

Activation of the B-cell specific transcription factor Bach2 by peroxide

Activation of the B-cell specific transcription factor Bach2 by peroxide

PU.1 and pRb Bind GATA-1 on DNA and Recruit a Histone H3K9 Methyl Transferase-Containing Complex to Repress the Erythroid Transcription Program.

Current work indicates that transcriptional repression is at least as important as transcriptional activation in normal development. Inappropriate or untimely transcriptional repression in immature hematopoietic cells is often the basis for a block to differentiation in hematologic malignancies. Activation of PU.1, a myeloid and B-cell specific transcription factor, in erythroid cells plays a key role in Friend virus-induced mouse erythroleukemia (MEL). Previous results from our laboratory showed that PU.1 blocks the erythroid differentiation-promoting activity of GATA-1 by binding directly to GATA-1 on DNA and inhibiting its transcriptional function. PU.1-mediated repression of GATA-1 on transiently transfected GATA-1 target genes is dependent on the corepressor pRb that also binds to PU.1 (Rekhtman et al., Genes & Dev 1999 and Mol Cell Biol 2003). To further investigate the mechanism of PU.1-mediated repression of GATA-1 in chromatin, we examined the occupancy of several GATA-1 target genes by PU.1 and pRb, as well as the state of core histone modifications at these loci in MEL cells by quantitative chromatin immunoprecipitation. These studies included both endogenous GATA-1 target genes and an exogenous GATA-1 target gene (alpha globin) integrated at a specific locus in MEL cells by Recombinase-Mediated Cassette Exchange. We found that GATA-1 sites at both the exogenous, integrated gene as well as at endogenous genes (including the regulatory regions of the alpha globin, beta globin, alas-e, eklf, p45 nf-e2) are occupied by a GATA1 - PU.1 - pRb complex in undifferentiated MEL cells. The presence of all three components of the complex is dependent on intact GATA-1 binding sites in the exogenous, integrated gene. The histone methyltransferase Suv39H1 and the histone H3MeK9 binding protein, HP1alpha, are also present at the repressed loci. During induced differentiation of MEL cells, PU.1, pRb, Suv39H1 and HP1alpha occupancy at these sites declines but GATA-1 continues to be present at its binding sites. The disruption of the repression complex at these loci during differentiation as well as during siRNA-mediated PU.1 knock down is associated with conversion of methylated H3K9 to acetylated H3K9 and significant transcriptional derepression of these GATA-1 target genes. These findings support a model for repression of GATA-1 by PU.1 at endogenous loci through recruitment of the corepressor pRb and associated histone methyltransferase (Suv39H1) and H3MeK9 binding (HP1alpha) activities.

New candidate loci for multiple sclerosis susceptibility revealed by a whole genome association screen in a Belgian population

We have completed a whole genome screen for association with multiple sclerosis (MS) in a Belgian population. The 6000 microsatellite markers provided through the Genetic Association of Multiple Sclerosis in EuropeanS (GAMES) collaborative were genotyped in case–control and family-based samples. The 20 most promising markers included three markers ( D6S1615, D6S2444 and TNFa) from the classically established HLA class II cluster and one ( D6S265) from the recently re-emphasized HLA class I cluster. In other highlighted regions, preliminary candidate genes from the immune system have been identified: e.g. the integrin ligand EDIL3, the high-mobility group box protein TOX, neutral sphingomyelinase activating factor (NSMAF) and the B-cell specific transcription factor POU2AF1.

BSAP/Pax5A Expression Blocks Survival and Expansion of Early Myeloid Cells Implicating Its Involvement in Maintaining Commitment to the B-Lymphocyte Lineage

Early B lymphopoiesis is marked by plasticity between the myeloid and B lineages. An attractive model for B-lineage development is that commitment to this lineage is partly determined by the ordered expression of genes that prohibit switching to the myeloid lineage. In this regard, whereas the role of the B-cell–specific transcription factor BSAP/Pax5A in regulating B-lymphoid–restricted gene expression has been well-established, its role in maintaining B-lineage commitment is unclear. Thus, BSAP/Pax5A was constitutively expressed in the multipotent EML cell line, which can be directed toward the myeloid lineage by culture with interleukin-3 (IL-3) and retinoic acid. EML cells expressing BSAP/Pax5A successfully acquired the myeloid lineage markers CD11b and F4/80 in response to IL-3 and retinoic acid, indicating differentiation to the myeloid lineage. However, these early myeloid cells failed to expand in culture with granulocyte-macrophage colony-stimulating factor and were directed instead toward an apoptotic pathway. In parallel, primary bone marrow stem cells transduced with retrovirus constitutively expressing BSAP/Pax5A began myeloid cell differentiation, but like the transformed EML model failed to expand in response to myeloid growth factors. These studies identify a role for BSAP/Pax5A in suppressing the response to myeloid growth factors, which may be a component of the regulatory processes that limit plasticity of early B-lymphoid progenitors.

BSAP/Pax5A Expression Blocks Survival and Expansion of Early Myeloid Cells Implicating Its Involvement in Maintaining Commitment to the B-Lymphocyte Lineage

AbstractEarly B lymphopoiesis is marked by plasticity between the myeloid and B lineages. An attractive model for B-lineage development is that commitment to this lineage is partly determined by the ordered expression of genes that prohibit switching to the myeloid lineage. In this regard, whereas the role of the B-cell–specific transcription factor BSAP/Pax5A in regulating B-lymphoid–restricted gene expression has been well-established, its role in maintaining B-lineage commitment is unclear. Thus, BSAP/Pax5A was constitutively expressed in the multipotent EML cell line, which can be directed toward the myeloid lineage by culture with interleukin-3 (IL-3) and retinoic acid. EML cells expressing BSAP/Pax5A successfully acquired the myeloid lineage markers CD11b and F4/80 in response to IL-3 and retinoic acid, indicating differentiation to the myeloid lineage. However, these early myeloid cells failed to expand in culture with granulocyte-macrophage colony-stimulating factor and were directed instead toward an apoptotic pathway. In parallel, primary bone marrow stem cells transduced with retrovirus constitutively expressing BSAP/Pax5A began myeloid cell differentiation, but like the transformed EML model failed to expand in response to myeloid growth factors. These studies identify a role for BSAP/Pax5A in suppressing the response to myeloid growth factors, which may be a component of the regulatory processes that limit plasticity of early B-lymphoid progenitors.

PU.1 regulates the expression of the human neutrophil elastase gene

PU.1 is a transcription factor present in B-cells and macrophages. Here, we report our studies on the role of PU.1 in myelopoiesis using human neutrophil elastase (HNE) as a model. HNE, a component of the primary granules of mature granulocytes, is a serine protease which is transcriptionally restricted to the late promyelocytic stage of granulocytic maturation. The first 200 bp of the HNE promoter directs myeloid specific expression of a reporter gene and a 30-bp element within this region was been identified as the major determinant of myeloid specific expression [S. Srikanth, T. Rado, A 30-bp element is responsible for the myeloid specific activity of the human neutrophil elastase promoter, J. Biol. Chem. 269 (1994) 32626–32632.]. We now show that the B-cell and macrophage specific transcription factor, PU.1, binds to the PU.1 consensus site within the 30-bp element to activate transcription. Substitution mutations within this recognition sequence results in the loss of PU.1 binding and in a 90% decrease in promoter activity in myeloid cells. Cotransfection of PU.1 and a reporter gene controlled by the HNE promoter into non-myeloid HeLa cells resulted in activation of reporter gene transcription.

Cooperation of Spi-1/PU.1 with an activated erythropoietin receptor inhibits apoptosis and Epo-dependent differentiation in primary erythroblasts and induces their Kit ligand-dependent proliferation.

Spi-1/PU.1 is a myeloid- and B-cell specific transcription factor which is also involved in Friend virus-induced murine erythroleukemia. The pre-leukemic phase of Friend erythroleukemia results from activation of the erythropoietin receptor (EpoR) by the spleen focus forming virus (SFFV) envelope glycoprotein, followed by the emergence of leukemic clones characterized by overexpression of Spi-1 and mutation of the p53 tumor suppressor gene. We developed a heterologous system to analyze the contribution of these alterations to the induction of primary erythroblast transformation. Avian erythroblasts expressing the activated mouse EpoR(R129C) differentiated into erythrocytes in response to hEpo. Expression of Spi-1 in these cells inhibited this ability to differentiate and rescued the cells from the apoptotic cell death program normally induced upon hEpo withdrawal. Although devoid of any effect by itself, a mutant p53 cooperated with Spi-1 and EpoR(R129C) to reinforce both phenotypes. Analysis of erythroblasts co-expressing Spi-1 and the wild-type mouse EpoR showed that differentiation arrest and inhibition of apoptosis depended on specific cooperation between Spi-1 and EpoR(R129C). This cooperation was also required to induce the sustained proliferation of differentiation-blocked erythroblasts in response to ligand activation of the endogenous tyrosine kinase receptor c-Kit. These results show that Spi-1/PU.1 requires signals emanating from specific cytokine and growth factor receptors to affect the survival, proliferation and differentiation control of primary erythroblasts. They also suggest that the function of Spi-1/PU.1 in the late phase of Friend leukemia requires specific signaling from the gp55-modified EpoR generated during the early phase of the disease.

The t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytoid lymphoma involves the PAX-5 gene

The t(9;14)(p13;q32) translocation is associated with approximately 50% of lymphoplasmacytoid lymphoma (LPL), a subtype of B-cell non-Hodgkin's lymphoma (NHL). We cloned the chromosomal breakpoint of der (14) from an LPL case (1052) and showed that it involved a junction between 9p13 and the switch micro region of the Ig heavy chain locus (IgH) on 14q32. Using a YAC contig spanning 1.5 megabase (Mb), we determined that the 9p13 breakpoint in one case (1052) mapped within a 270-kb restriction fragment containing two previously reported 9p breakpoints associated with a alpha-heavy chain disease case (MAL) and KI-1 positive diffuse large cell lymphoma (DLCL) cell line (KIS-1). The same fragment also contained the PAX-5 gene which encodes a B-cell specific transcription factor involved in the control of B-cell proliferation and differentiation. The breakpoints of KIS-1 and 1052 were mapped within the 5' noncoding region of PAX-5, while the 9p13 breakpoint of MAL mapped 230 to 270 kb upstream to PAX-5. In all three cases, the translocation caused the juxtaposition of the PAX-5 gene to the IgH locus in the opposite direction of transcription. When compared with six other DLCL cell lines lacking t(9;14)(p13;q32), the KIS-1 cell line showed an 11-fold overexpression of PAX-5 mRNA and a significantly reduced expression of the p53 gene, which is normally regulated by PAX-5. Moreover, metaphase and interphase fluorescence in situ hybridization (FISH) analysis using a YAC clone spanning 1 Mb including the PAX-5 as a probe identified chromosomal translocations in 5 of 7 cases carrying 9p13 translocations. These findings suggest that the PAX-5 gene is the target of the t(9;14) in LPL whereby its expression may be deregulated by juxtaposition to IgH regulatory elements, thus contributing to lymphomagenesis.

The t(9; 14)(p13; q32) Chromosomal Translocation Associated With Lymphoplasmacytoid Lymphoma Involves the PAX-5 Gene

AbstractThe t(9; 14)(p13;q32) translocation is associated with approximately 50% of lymphoplasmacytoid lymphoma (LPL), a subtype of B-cell non-Hodgkin’s lymphoma (NHL). We cloned the chromosomal breakpoint of der (14) from an LPL case (1052) and showed that it involved a junction between 9p13 and the switch µ region of the Ig heavy chain locus (IgH) on 14q32. Using a YAC contig spanning 1.5 megabase (Mb), we determined that the 9p13 breakpoint in one case (1052) mapped within a 270-kb restriction fragment containing two previously reported 9p breakpoints associated with a α-heavy chain disease case (MAL) and Ki-1 positive diffuse large cell lymphoma (DLCL) cell line (KIS-1). The same fragment also contained the PAX-5 gene which encodes a B-cell specific transcription factor involved in the control of B-cell proliferation and differentiation. The breakpoints of KIS-1 and 1052 were mapped within the 5′ noncoding region of PAX-5, while the 9p13 breakpoint of MAL mapped 230 to 270 kb upstream to PAX-5. In all three cases, the translocation caused the juxtaposition of the PAX-5 gene to the IgH locus in the opposite direction of transcription. When compared with six other DLCL cell lines lacking t(9; 14)(p13;q32), the KIS-1 cell line showed an 11-fold overexpression of PAX-5 mRIMA and a significantly reduced expression of the p53 gene, which is normally regulated by PAX-5. Moreover, metaphase and interphase fluorescence in situ hybridization (FISH) analysis using a YAC clone spanning 1 Mb including the PAX-5 as a probe identified chromosomal translocations in 5 of 7 cases carrying 9p13 translocations. These findings suggest that the PAX-5 gene is the target of the t(9; 14) in LPL whereby its expression may be deregulated by juxtaposition to IgH regulatory elements, thus contributing to lymphomagenesis.

Solution structure of a POU-specific homeodomain: 3D-NMR studies of human B-cell transcription factor Oct-2.

The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression. This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD). Here we describe the solution structure of a POU-specific homeodomain. An NMR model is obtained from Oct-2, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes. A fragment of Oct-2 containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy. Complete 1H and 15N resonance assignment of the POUHD moiety is presented. The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains. A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution. Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs.

Extinction of Ig genes expression in myeloma x fibroblast somatic cell hybrids is accompanied by repression of the oct-2 gene encoding a B-cell specific transcription factor.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of differentiation-specific traits of the nonfibroblast parental cell. To explore the genetic basis of this phenomenon, we have used a series of somatic cell hybrids between myeloma cells and fibroblasts. Previous findings show that in these hybrids expression of the immunoglobulin (Ig) genes was extinguished at the transcriptional level. Our present results show that NF-kappa B transcription factor, known to be critical for kappa-chain enhancer activity, is present although in a lower amount, in the nucleus and in the cytosolic fraction of most of these hybrids (probably attached to the previously postulated I-kappa B inhibitor). In contrast, the expression of the NF-A2/OTF-2 transcription factor encoded by the oct-2 gene, which binds to the octameric motif located in the Ig promoters and heavy chain gene enhancer, is extinguished at the transcriptional level. Our data thus suggest that extinction of Ig genes expression occurs via an indirect mechanism in which a fibroblast factor suppresses transcription factor(s) which are critical for Ig transcription.