Zbynek Kozmik

The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP.

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 beta-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.

Independent regulation of the two Pax5 alleles during B-cell development

The developmental control genes of the Pax family are frequently associated with mouse mutants and human disease syndromes. The function of these transcription factors is sensitive to gene dosage, as mutation of one allele or a modest increase in gene number results in phenotypic abnormalities. Pax5 has an important role in B-cell and midbrain development. By following the expression of individual Pax5 alleles at the single-cell level, we demonstrate here that Pax5 is subject to allele-specific regulation during B-lymphopoiesis. Pax5 is predominantly transcribed from only one allele in early progenitors and mature B cells, whereas it switches to a biallelic transcription mode in immature B cells. The allele-specific regulation of Pax5 is stochastic, reversible, independent of parental origin and correlates with synchronous replication, in contrast with imprinted and other monoallelically expressed genes. As a consequence, B-lymphoid tissues are mosaics with respect to the transcribed Pax5 allele, and thus mutation of one allele in heterozygous mice results in deletion of the cell population expressing the mutant allele due to loss of Pax5 function at the single-cell level. Similar allele-specific regulation may be a common mechanism causing the haploinsufficiency and frequent association of other Pax genes with human disease.

Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8

Transcription factors of the Pax family bind to their target genes via the paired domain which is known to be composed of two subdomains each recognizing distinct half‐sites in adjacent major grooves of the DNA helix. We now demonstrate that the mammalian Pax8 gene gives rise, by alternative mRNA splicing, to a protein isoform containing an extra serine residue in the recognition α‐helix 3 of the paired domain. This Pax8(S) protein does not interact with bipartite paired domain‐binding sites, indicating that inactivation of the N‐terminal DNA‐binding motif severely restricts the sequence specificity of the paired domain. However, the Pax8(S) protein binds in vitro and in vivo to the 5aCON sequence which was previously identified as a high‐affinity binding site for the Pax6(5a) splice variant carrying a 14‐amino‐acid insertion in the paired domain. The 5aCON sequence is shown to consist of four interdigitated 5′ half‐sites of the bipartite consensus sequence and is thus bound by four Pax8(S) molecules via the intact C‐terminal DNA‐binding motif of the paired domain. Together these data suggest that inactivation of the N‐terminal region of the paired domain by alternative splicing is used in vivo to selectively target Pax transcription factors to gene regulatory regions containing highly specialized 5aCON‐like sequences.

Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different transactivation properties.

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 Pax-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 Pax-8 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 of Pax-8 gene transcripts not only generates six different Pax-8 variants but is also temporally and spatially regulated during early mouse development.

The characterization of novel Pax genes of the sea urchin and Drosophila reveal an ancient evolutionary origin of the Pax2/5/8 subfamily

The developmental control genes of the Pax family can be grouped into different subclasses according to structure and sequence homology. Here we describe the isolation and characterization of three novel Pax genes of the sea urchin for which no homologues are yet known in other animal phyla. One of these genes, suPaxB, codes for the previously characterized transcription factor TSAP which is involved in the developmental regulation of two pairs of late histone genes. Furthermore, conserved members of the Pax2/5/8 subfamily, which have so far been described only in vertebrates, were isolated not only from the sea urchin, but also from Drosophila and C. elegans. Hence, the Pax2/5/8 transcription factors constitute an ancient subfamily of highly conserved Pax proteins. During Drosophila embryogenesis, the Pax258 gene is shown to be expressed in the precursor cells of the external sensory organs, thus suggesting a role for Pax258 in the early development of the peripheral nervous system of insects.

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

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

Non-essential role for cilia in coordinating precise alignment of lens fibres.

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet-Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP.

The gene coding for the B cell surface protein CD19 is localized on human chromosome 16p11

The CD19 gene codes for one of the earliest markers of the human B cell lineage and is a target for the B lymphoid-specific transcription factor BSAP (Pax-5). The transmembrane protein CD19 has been implicated in controlling proliferation of mature B lymphocytes by modulating signal transduction through the antigen receptor. In this study, we have employed Southern blot and fluorescence in situ hybridization analyses to localize the CD19 gene to human chromosome 16p11.