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EVOLUTIONARY HOMOLOGS part 4/5

Pax5 mutation

The Pax5 gene, coding for the transcription factor BSAP, was mutated in the mouse germline by targeted disruption. Homozygous mutant mice are born alive, became growth retarded, and usually die within three weeks. About 5% of mutants survive to adulthood and are fertile, but severely runted. Morphogenesis of the posterior midbrain is affected as early as embryonic day 16.5, leading to a reduction of the inferior colliculus near the midline and to altered foliation of the anterior cerebellum. All mutants fail to produce small pre-B, B, and plasma cells, owing to a complete arrest of B cell development at an early precursor stage. These data define a key role for Pax5 in early B lymphopoiesis and midbrain patterning (Urbanek, 1994).

Pax5 protein interactions

Pax-5 codes for the transcription factor BSAP, which plays an important role in midbrain patterning, B cell development, and lymphoma formation. Pax-5 is known to control gene expression by recognizing its target genes via the NH2-terminal paired domain and by regulating transcription through a COOH-terminal regulatory module consisting of activating and inhibitory sequences. The central region of Pax-5 contains a sequence with significant homology to the first alpha-helix of the paired-type homeodomain. This partial homeodomain has been highly conserved throughout vertebrate evolution because it is found not only in Pax-5 but also in the related Pax-2 and Pax-8 members of the same Pax subfamily. The partial homeodomain binds the TATA-binding protein (TBP) and retinoblastoma (Rb) gene product. Both TBP and Rb were shown by coimmunoprecipitation experiments to directly associate with Pax-5 in vivo. The conserved core domain of TBP and the pocket region as well as COOH-terminal sequences of Rb are required for interaction with the partial homeodomain of Pax-5 in in vitro binding assays. Furthermore, Pax-5 is specifically bound only by the underphosphorylated form of Rb. These data indicate that Pax-5 is able to contact the basal transcription machinery through the TBP-containing initiation factor TFIID, and that its activity can be controlled by the cell cycle-regulated association with Rb (Eberhard, 1999).

Pax5 regulation

Pax and homeodomain transcription factors are essential for the formation of an organizing center at the midbrain-hindbrain boundary (mhb), which controls the genesis of the midbrain and cerebellum in the vertebrate embryo. Pax2 and Pax5 are sequentially activated in this brain region, with Pax2 expression preceding that of Pax5. Using a transgenic reporter assay, a conserved 435 bp enhancer has been identified in the 5' flanking region of mammalian Pax5 genes that directs lacZ expression in the correct temporal and spatial pattern at the mhb. This minimal enhancer is composed of two distinct elements, as shown by protein-binding assays with mhb-specific extracts. The proximal element contains overlapping consensus binding sites for members of the Pax2/5/8 and POU protein families, whereas a distal element is bound by homeodomain and zinc finger transcription factors. Expression analysis of transgenes carrying specific mutations in these recognition motifs has identified the Pax- and homeodomain-binding sites as functional elements that cooperatively control the activity of the mhb enhancer. lacZ genes under the control of either the minimal enhancer or the endogenous Pax5 locus are normally expressed at the mhb in Pax5 mutant embryos, indicating that this enhancer does not depend on autoregulation by Pax5. However, in Pax2 mutant embryos, expression of the endogenous Pax5 gene is delayed and severely reduced in lateral aspects of the neural plate, which upon neural tube closure, becomes the dorsal mhb region. This cross-regulation by Pax2 is mediated by the Pax-binding site of the minimal enhancer: upon specific mutation, this results in severely reduced transgene expression in the dorsal part of the mhb. Together these data indicate that Pax2 and homeodomain proteins directly bind to and cooperatively regulate the mhb enhancer of Pax5 (Pfeffer, 2000).

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address the genetic pathways through which FGF8 acts, the epistatic relationships of mid/hindbrain genes that respond to FGF8 were examined, using a novel mouse brain explant culture system. En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, it was found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone (Liu, 2001).

Pax5 and lymphoid development

The transcription factor Pax-5 is expressed during the early stages of B-cell differentiation and influences the expression of several B-cell-specific genes. In addition to the existing isoform (Pax-5, which has been renamed Pax-5a), three new isoforms, Pax-5b, Pax-5d, and Pax-5e, have been isolated from murine spleen and B-lymphoid cell lines using library screenings and polymerase chain reaction amplification. Isoforms Pax-5b and Pax-5e have their second exon spliced out, resulting in proteins with only a partial DNA-binding domain. Isoforms Pax-5d and Pax-5e have deleted the 3'-region, which encodes the transactivating domain, and replaced it with a novel sequence. Pax-5a and Pax-5b proteins have been detected using Western blot analysis. Pax-5a is detectable in pro-, pre-, and mature B-cell lines, but not in two plasmacytomas; Pax-5b is present at low levels in mature B-cell lines and, unexpectedly, in one plasma cell line, but not in pro-B-cell or T-cell lines. In vitro translated Pax-5a and Pax-5d, but not Pax-5b or Pax-5e, can interact with a B-cell-specific activator protein-binding site on the blk promoter. Pax-5d is present in nuclear extracts of some (but not all) B-lymphoid lines and interacts with the B-cell-specific activator protein-binding site. The pattern of differential expression of alternatively spliced Pax-5 isoforms suggests that they may be important regulators of transcription during B-cell maturation (Zwollo, 1997)

The Pax5 gene coding for the transcription factor BSAP has an essential role in B lymphopoiesis and midbrain development. A detailed analysis of the B-cell phenotype of Pax5 mutant mice is presented that reveals a differential dependency of fetal and adult B lymphopoiesis on this transcriptional regulator. B-cell development is arrested in the bone marrow at the early pro-B (pre-BI) cell stage, which is characterized by expression of the early markers c-kit, CD43, lambda5, VpreB, and HSA and the absence of later markers CD25 and BP-1. These pre-BI cells fail to express the BSAP target gene CD19 and are capable of long-term proliferation in vitro in the presence of stromal cells and IL-7. B-lymphoid progenitors can not be detected in the fetal liver of Pax5 mutant embryos. However, Pax5-deficient fetal liver cells give rise to the development of pre-BI cells in bone marrow on transplantation into lethally irradiated mice. These data indicate different functions of Pax5 in the distinctive microenvironments of fetal liver and adult bone marrow. As shown by PCR analyses, the pre-BI cells in Pax5-deficient bone marrow undergo D(H)-to-J(H) rearrangement of the immunoglobulin heavy-chain locus at normal frequency. In contrast, V(H)-to-D(H)J(H) rearrangements are reduced approximately 50-fold in Pax5-deficient pre-BI cells, suggesting a role for Pax5 in the developmental pathway controlling V-to-DJ recombination (Nutt, 1997).

The paired box transcription factor Pax-5 (B-cell-specific activator protein) is a key regulator of lineage-specific gene expression and differentiation in B-lymphocytes. Pax-5 functions as a cell type-specific docking protein that facilitates binding of the early B-cell-specific mb-1 promoter by proteins of the Ets proto-oncogene family. Transcriptional activity of the mb-1 promoter in pre-B-cells is critically dependent on binding sites for Pax-5:Ets complexes. Ternary complex assembly requires only the Pax-5 paired box and ETS DNA-binding domains. Mutation of a single base pair in the ternary complex binding site allows for independent binding by Ets proteins but, conversely, inhibits the binding of Pax-5 by itself. Strikingly, the mutation reverses the pattern of complex assembly: Ets proteins recruit Pax-5 to bind the mutated sequence. Recruitment of Net and Elk-1 (but not SAP1a) by Pax-5 defines a functional difference between closely related Ets proteins. Replacement of a valine (V68) in the ETS domain of SAP1a by aspartic acid (as found in c-Ets-1, Elk-1, and Net) enhances ternary complex formation by more than 60-fold. Together, these observations define novel transcription factor interactions that regulate gene expression in B cells (Fitzsimmons, 1996).

During B cell differentiation, the pre-B cell stage plays a significant role in immunoglobulin gene rearrangement in the context of the allelic exclusion and kappa chain gene rearrangement. The early B cell-specific binding protein (EBB)-1 transcription factor binds to the promoters of two pre-B cell-specific genes, VpreB and lambda5, and regulates their pre-B cell-specific expression. EBB-1 binds to the KI and KII sites in the upstream area of Jkappa region, which are crucial for kappa chain gene rearrangement. Gene transfer and gel-shift assays demonstrate that EBB-1 is identical to Pax-5 and binds to promoters of VpreB and lambda5 as well as the KI and KII sites. These results suggest that Pax-5 plays an important role in the coordinate regulation of several immunoglobulin gene family members that are crucial in B cell development (Tian, 1997).

Cytokine regulation of B cell development was analyzed using interleukin-2 (IL-2)-induced transcription of the J chain gene as a model system. A nuclear target of the IL-2 signal is identified as the Pax5 transcription factor, BSAP, which recognizes a negative regulatory motif in the J chain promoter. Functional assays show that BSAP mediates the silencing of the J chain gene during the early stages of B cell development, but repression is relieved during the antigen-driven stages in a concentration-dependent manner by an IL-2-induced down-regulation of BSAP RNA expression. At the low levels present in J chain-expressing plasma cells, BSAP repression can be overridden by positive-acting factors binding to down-stream J chain promoter elements. Overexpression of BSAP in these cells reverses the positive regulation and inhibits J chain gene transcription. Thus, IL-2 regulation of BSAP concentration may provide a mechanism for controlling both repressor and activator functions of BSAP during a B cell immune response (Rinkenberger, 1996).

The Pax5 transcription factor BSAP (B-cell-specific activator protein) is known to bind to and repress the activity of the immunoglobulin heavy chain 3' alpha enhancer. An element, designated alpha P, lies approximately 50 bp downstream of the BSAP binding site 1 and is required for maximal enhancer activity. In vitro binding experiments suggest that the 40-kDa protein that binds to this element (NF-alpha P) is a member of the Ets family present in both B-cell and plasma-cell nuclei. However, in vivo footprint analysis suggests that the alpha P site is occupied only in plasma cells, whereas the BSAP site is occupied in B cells but not in plasma cells. When Pax5 binding to the enhancer in B cells is blocked in vivo by transfection with a triple-helix-forming oligonucleotide, an alpha P footprint appears and endogenous immunoglobulin heavy chain transcripts increase. The triple-helix-forming oligonucleotide also increases enhancer activity of a transfected construct in B cells, but only when the alpha P site is intact. Pax5 thus regulates the 3' alpha enhancer and immunoglobulin gene transcription by blocking activation by NF-alpha P (Neurath, 1995).

Pax5, lymphoma and Medulloblastoma

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). The chromosomal breakpoint involves a junction between 9p13 and the switch micro region of the Ig heavy chain locus (IgH) on 14q32. The 9p13 breakpoint in one case maps within a 270-kb restriction fragment containing breakpoints associated with an alpha-heavy chain disease case (MAL) and KI-1 positive diffuse large cell lymphoma (DLCL) cell line (KIS-1). The same fragment also contains the PAX-5 gene, which encodes a B-cell specific transcription factor involved in the control of B-cell proliferation and differentiation. The breakpoints map within the 5' noncoding region of PAX-5, while the 9p13 breakpoint of MAL maps 230 to 270 kb upstream of PAX-5. In all three cases, the translocation causes the juxtaposition of the PAX-5 gene to the IgH locus in the opposite direction of transcription. Cell lines containing the translocation show an 11-fold overexpression of PAX-5 mRNA and a significantly reduced expression of the p53 gene, which is normally regulated by PAX-5. 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 (Iida, 1996).

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene reveal that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A t(9;14) breakpoint justaposes the immunoglobulin heavy-chain (IgH) locus on 14q32 to chromosome 9p13. The translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity to the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation (Busslinger, 1996).

Medulloblastoma is a pediatric brain tumor originating in the human cerebellum. A collection of 23 medulloblastomas was analyzed for expression of the developmental control genes of the PAX and EN gene families. Of all nine PAX genes investigated, only PAX5 and PAX6 (Drosophila homolog: Eyeless) are consistently expressed in most medulloblastomas (70 and 78% of all cases, respectively), as are the genes EN1 (57%) and EN2 (78%) (See Drosophila Engrailed). EN1, EN2, and PAX6 genes are also expressed in normal cerebellar tissue, and their expression in medulloblastoma is consistent with the hypothesis that this tumor originates in the external granular layer of the developing cerebellum. PAX5 transcripts are, however, not detected in the neonatal cerebellum, indicating that this gene is deregulated in medulloblastoma. In the desmoplastic variant of medulloblastoma, PAX5 expression is restricted to the reticulin-producing proliferating tumor areas containing undifferentiated cells; PAX5 is not expressed in the reticulin-free nonproliferating islands undergoing neuronal differentiation. These data suggest that deregulated expression of PAX5 correlates positively with cell proliferation and inversely with neuronal differentiation in desmoplastic medulloblastoma (Kozmik, 1995).

Pax8 gene structure and alternative splicing

Evolutionary homologs continued: part 5/5 | back to part 1/5 | part 2/5 | part 3/5


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