BSAP has been identified previously as a transcription factor that is expressed at early, but not late, stages of B-cell differentiation. Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins. BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse. An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP. Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1. During embryogenesis, the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system (CNS). Later, the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis. BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse. All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis.

The Pax-5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre-BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre-BI cells of wild-type and Pax-5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax-5-deficient pre-BI cells by a hormone-inducible BSAP-estrogen receptor fusion protein. The genes coding for the B-cell receptor component Ig-alpha (mb-1) and the transcription factors N-myc and LEF-1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD-1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax-5-deficient pre-BI cells with full-length BSAP or a truncated form containing only the paired domain. IL-7 signalling was able to efficiently induce the N-myc gene only in the presence of full-length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb-1 and LEF-1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA-binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss- and gain-of-function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context.

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a f-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.The differentiation of B lymphocytes from progenitor cells to immunoglobulin-secreting plasma cells is a multistep process which is characterized by the sequential expression of specific cell surface markers involved in cell-cell interaction and signal transduction (29). Among them is the B-cellspecific antigen CD19. This protein is expressed throughout B-cell ontogeny from the early progenitor cell up to the mature B-cell stage and is lost from the cell surface only at the terminal stages of B-cell differentiation. The CD19 molecule is present on both normal and malignant B-lymphoid cells and hence is the most reliable diagnostic surface marker of the human B-cell lineage (23, 35). The CD19 gene codes for a 95-kDa protein with two extracellular immunoglobulinlike domains, a short transmembrane region, and a large cytoplasmic tail (33,34,38). These features suggest that the CD19 protein is a membrane receptor involved in signal transduction of B lymphocytes. No natural ligand for CD19 is as yet known; instead, monoclonal anti-CD19 antibodies have been used to study the physiological function of the CD19 molecule. In agreement with the receptor hypothesis, antibody binding leads to rapid internalization of CD19 (7,35) and to mobilization of free intracellular calcium ions (17,27,35). CD19 antibodies strongly inhibit proliferation of resting mature B cells in response to stimulation with antiimmunoglobulin M (IgM) antibodies (11,27,35 (14).Regulation of B-cell-specific gene expression is brought about by several distinct transcription factors (reference 3 and references therein). One of them is the B-cell lineagespecific activator protein (BSAP). We have identified this transcription factor as a mammalian homolog of the sea urchin protein TSAP, which is res...

Pax‐5 encodes the transcription factor BSAP which plays an essential role in early B cell development and midbrain patterning. In this study we have analysed the structural requirements for transcriptional activation by BSAP. In vitro mutagenesis and transient transfection experiments indicate that the C‐terminal serine/threonine/proline‐rich region of BSAP contains a potent transactivation domain of 55 amino acids which is active from promoter and enhancer positions. This transactivation domain was found to be inactivated by a naturally occurring frameshift mutation in one PAX‐5 allele of the acute lymphoblastic leukemia cell line REH. The function of the transactivation domain is negatively regulated by adjacent sequences from the extreme C‐terminus. The activating and inhibitory domains function together as an independent regulatory module in different cell types as shown by fusion to the GAL4 DNA binding domain. The same arrangement of positively and negatively acting sequences has been conserved in the mammalian Pax‐2 and Pax‐8, the zebrafish Pax‐b as well as the sea urchin Pax‐258 proteins. These data demonstrate that the transcriptional competence of a subfamily of Pax proteins is determined by a C‐terminal regulatory module composed of activating and inhibitory sequences.

Pax-8, a member of the paired box-containing gene family, was shown to be coexpressed with Pax-2 in several human kidney carcinoma cell lines. Four different Pax-8 mRNA isoforms, a to d, were cloned from one of these cell lines by polymerase chain reaction amplification, and the Pax-8 gene was isolated from a human cosmid library. Analysis of the exon-intron structure of Par-8 revealed that the four mRNA isoforms arise by alternative splicing, resulting in inclusion or exclusion of exon 7 and/or exon 8 sequences. All four proteins retain the paired domain as their DNA-binding motif and recognize DNA in the same manner as do the closely related Pax-2 and BSAP (Pax-5) proteins. The Pax-8a and Pax-8b isoforms end in a serine/threonine/ tyrosine-rich sequence, while the C terminus of Pax-8c and Pax-8d is translated in a different, proline-rich reading frame. Transient transfection experiments revealed that Pax-8 isoforms a and b, but not c and d, strongly stimulate transcription from a promoter containing six copies of a paired-domain recognition sequence. The same four mRNA variants were also detected by RNase protection analysis in the mouse embryo and adult kidney, thus indicating evolutionary conservation of Pax-8 mRNA splicing. A different splice pattern was observed in the developing placenta, which expresses two new variants, Pax-8e and Pax-8f, instead of transcripts b to d. Expression of these mRNAs is high at embryonic day 9.5 and is gradually reduced until Pax-8a is the predominant transcript in the 12.5-day placenta. In the embryo, however, the synthesis of mRNAs b to d is initially low and then increases relative to that of Pax-8a. Hence, alternative splicing ofPax-8 gene transcripts not only generates six different Pax-8 variants but is also temporally and spatially regulated during early mouse development.