Pax8 gene structure and alternative splicing

Lambda phage clones containing the murine Pax 8 gene were isolated from a C57BL/6 kidney genomic mouse library using mouse cDNA fragments as probes. A clone encompassing about 16 kb of the 5' untranslated region of the murine Pax 8 gene was isolated from a mouse embryonic stem cell (D3) library. The murine Pax 8 gene has a size of approximately 26 kb and contains the coding sequence for mRNA in 12 exons. The major and several minor transcription initiation sites were identified. Position +1 is located 488 nucleotides upstream of the ATG initiation codon and 24 bases downstream of a TATA-like sequence, ATAAAA. The translation initiation and termination sites are located in exons 2 and 12, respectively. Further analysis of 570 bases of the 5' flanking sequence reveal AP2, SP1, PEA3, zeste, NF-kappaB, and CCAAT consensus binding sites. Ribonuclease protection assays with a probe spanning the first two exons of mouse Pax 8 cDNA on total RNA samples isolated from different tissues of newborn mice show that the murine Pax 8 gene is predominantly expressed in kidney tissue. Low levels of Pax 8 gene expression are also found in the liver, spleen, lung, brain, and heart. The same transcription initiation sites are utilized in different tissues of newborn mice and embryo at Day 10.5 postconception. The murine Pax 8 gene is located on chromosome 2, map position B (Okladnova, 1997).

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. The mammalian Pax8 gene gives rise, by alternative mRNA splicing, to a protein isoform containing an extra serine residue in the recognition alpha-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 is a high-affinity binding site for the Pax6(5a) splice variant carrying a 14-amino-acid insertion in the paired domain. The 5aCON sequence consists 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 (Kozmik, 1997).

Pax8 phosphorylation

The conserved structure of the transcription factors of the Pax gene family may reflect functional conservation. The human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localization, in addition to an activating region at the carboxy terminus of the protein and an inhibitory region encoded by the exon 9, present only in a splice variant PAX8a. Regions of PAX8 determining the nuclear localization of the PAX8A/lacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS). These NLS have been identified in the paired domain and between the octapeptide and the residual homeodomain, respectively. The activating domain is encoded by the exons 10 and 11 and its function is modulated by the adjacent domains encoded by the exons 9 and 12. The domain encoded by exon 9 significantly inhibits the function of the activating domain. Pax8 is expressed in thyroid cells and its product binds promoters of the thyroglobulin and thyroperoxidase genes through its paired domain. Thyroid cell growth and differentiation depend on thyrotropin; by stimulating cAMP synthesis, thyrotropin activates the cAMP-dependent protein kinase A (See Drosophila PKA). A link between thyrotropin stimulation and gene activation by Pax8 has been investigated. Stimulation of cAMP synthesis augments Pax8-specific transcription in thyroid cells, indicating that PKA is involved in Pax8 activation. Cotransfection of GAL4/PAX8 fusions and the catalytic subunit of PKA in A126, a PKA-deficient derivative of the PC12 pheochromocytoma cell line, synergistically activates the GAL4-specific reporter, suggesting the activating domain of PAX8 is dependent upon the catalytic subunit of the PKA. It is proposed that this dependence is due to a hypothetical adaptor, which forms a target for PKA and interacts with the activating domain of PAX8. PAX8 isolated from the thyroid cell line FTRL5 is a phosphoprotein in which phosphorylation is not dependent on cAMP pathway activation. These results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotropin-dependent gene activation in thyroid cells. Investigation of PAX8 expression in a panel of Wilms' tumors shows a striking correlation between the expression of PAX8 and another transcription factor, WT1, indicating that these two genes may interact in vivo (Poleev, 1997).

Pax8 miscellaneous targets

The bcl-2 proto-oncogene suppresses apoptosis in a variety of cell types and is essential for normal renal development. PAX8 is a member of the paired box class of transcription factors and is developmentally regulated. bcl-2 and PAX8 are both expressed in the kidney during development and throughout life, demonstrating a nearly identical pattern of expression. Transient transfection reporter assays and electrophoretic mobility shift assays were used to test PAX8 transcriptional regulation of bcl-2. PAX8 transcriptionally activates the bcl-2 promoter and binds to promoter sequences in vitro. These findings establish that PAX8 can transcriptionally regulate bcl-2 and suggest that suppression of apoptosis is an important event in the development and maintenance of renal tubular epithelial structures (Hewitt, 1997).

Direct interactions between the genes that regulate development and those that regulate the cell cycle would provide a mechanism by which numerous biological events could be better understood. PAX5 plays a direct role in the control of p53 transcription. In primary human diffuse astrocytomas, PAX5 expression inversely correlates with p53 expression. The human p53 gene harbours a PAX binding site within its untranslated first exon that is conserved throughout evolution. PAX5 and its paralogues PAX2 and PAX8 are capable of inhibiting both the p53 promoter and transactivation of a p53-responsive reporter in cell culture. Mutation of the identified binding site eliminates PAX protein binding in vitro and renders the promoter inactive in cells. These data suggest that PAX proteins might regulate p53 expression during development and propose a novel alternative mechanism for tumour initiation or progression, by which loss of p53 function occurs at the transcriptional level (Stuart, 1995).

The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). An enhancer is located between nucleotides -2264 and -2495 in the 5'-flanking region of the NIS gene that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer (NUE) stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site (Ohno, 1999).

Zebrafish pax8 is required for otic placode induction and plays a redundant role with Pax2 genes in the maintenance of the otic placode

Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8, the entire gene, including the 5' and 3' UTRs, was sequenced. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8, or another early regulator, dlx3b, which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1, which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8. Disrupting pax8, pax2a and pax2b did not further impair otic induction relative to loss of pax8 alone. However, the amount of otic tissue gradually decreased in pax8-pax2a-pax2b-deficient embryos such that no otic tissue was detectable by 24 hours post-fertilization. Loss of otic tissue did not correlate with increased cell death, suggesting that otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain otic fate (Mackereth, 2005).

Pax8, renal development and Wilms' tumor gene

Pax genes encode a family of highly conserved DNA-binding transcription factors. These proteins play key roles in regulating a number of vertebrate and invertebrate developmental processes. Mutations in Pax-6 result in eye defects in flies, mice, and humans, and ectopic expression of this gene can trigger the development of ectopic compound eyes in flies. Likewise, mutation of other Pax genes in vertebrates results in the failure of specific differentiation programs; Pax-1 causes skeletal defects; Pax-2, kidney defects; Pax-3 or Pax-7, neural crest defects; Pax-4, pancreatic beta-cell defects; Pax-5, B-cell defects; Pax-8, thyroid defects; and Pax-9, tooth defects. Although this class of genes is obviously required for the normal differentiation of a number of distinct organ systems, members of this class have not previously been demonstrated to be capable of directing the embryonic development of organs in vertebrates. In this report, it is demonstrated that Pax-8 plays such a role in the establishment of the Xenopus embryonic kidney, the pronephros. However, in order to efficiently direct cells to form pronephric kidneys, XPax-8 requires cofactors, one of which may be the homeobox transcription factor Xlim-1. These two genes are initially expressed in overlapping domains in late gastrulae, and cells expressing both genes will go on to form the kidney. Ectopic expression of either gene alone has a moderate effect on pronephric patterning, while coexpression of XPax-8 plus Xlim-1 results in the development of embryonic kidneys of up to five times normal complexity and also leads to the development of ectopic pronephric tubules. This effect is synergistic rather than additive. XPax-2 can also synergize with Xlim-1, but the expression profile of this gene indicates that it normally functions later in pronephric development than does XPax-8. Together these data indicate that the interaction between XPax-8 and Xlim-1 is a key early step in the establishment of the pronephric primordium (Carroll, 1999).

Recent evidence indicates a crucial role for paired box genes in mouse and human embryogenesis. The murine Pax8 gene encodes a sequence-specific transcription factor and is expressed in the developing secretory system as well as in the developing and adult thyroid. This restricted expression pattern suggests involvement of the Pax8 gene in the morphogenesis of the above organs and prompted an investigation of the PAX8 gene in humans. An open reading frame of 450 amino acids contains the 128 amino acid paired domain at its amino-terminal end. The predicted human and mouse Pax8 proteins show 97.8% conservation and are identical in their paired domains. Two independent cDNA clones reveal differential splicing of the PAX8 transcripts,resulting in the removal of a 63 amino acid serine-rich region from the carboxy end of the predicted Pax8 protein. The truncated Pax8 protein becomes more similar to the predicted murine Pax2 protein, which is also expressed during kidney development and lacks the serine rich region. Both PAX8 transcripts are present in human thyroid, kidney and five Wilms' tumors. No truncated Pax8 transcripts could be detected in mouse kidney. In situ hybridization to sections of human embryonic and fetal kidney show expression of PAX8 in condensed mesenchyme, and comma- and S-shaped bodies. In contrast, PAX2 expression is present mainly in the very early stages of differentiation, in the induced, condensing mesenchyme. This restricted expression pattern suggests a specific role for both genes during glomeruli maturation (Poleev, 1992).

The Wilms' tumor gene (WT1) is an essential gene for kidney and gonadal development, although how WT1 expression is induced in these tissues is not known. One kidney transcription factor likely to play a role in this regulation is PAX 8. The co-expression of WT1 and PAX 8 during kidney development and in Wilms' tumors with an epithelium predominant histology suggests a possible interaction, and indeed, potential core PAX-binding sites have been identified in the WT1 promoter. Endogenous PAX 8 plays an important role in the activation of the WT1 promoter, since promoter activity is much stronger in cells with PAX 8 than in those without. Using binding assays, evidence was sought of PAX 8-DNA interactions throughout the 652-base pair human WT1 promoter: only one functional PAX 8 site with DNA binding activity was found, located 250 base pairs 5' of the minimal promoter. The responsiveness of the PAX 8 site was confirmed by assessing its ability to function as an enhancer significantly activating the minimal promoter in a position- and orientation-independent manner. Either endogenous or exogenously added PAX 8 activates the WT1 promoter; this promoter up-regulation depends upon the presence of an intact PAX 8-binding site. In contrast, the previously reported core PAX 8-binding sites identified by computer analysis of the WT1 promoter fail to specifically bind in vitro translated PAX 8 protein or activate the minimal promoter. Thus, a novel functional binding site for the transcription factor PAX 8 has been identified, suggesting that part of its role in kidney development may be as a modulator of WT1 expression in the kidney (Fraizer, 1997).

The developing renal system has long been exploited to study the regulation of gene expression during mesenchymal-epithelial transitions. Several transcription factors, including WT1 and PAX8, are expressed early in nephrogenesis and play a key role in this process. The expression of PAX8 occurs in the induced mesenchyme of the developing kidney prior to the upregulation of WT1 levels in the same cells. In this report, an assesment was carried out to detrmine if the Pax-8 gene product resides upstream of wt1 in a common regulatory pathway. Transfection studies, as well as gel-shift assays, indicate that PAX8 transactivates wt1 through elements within a 38 bp conserved motif, present in human and murine promoters. Two PAX8 isoforms, generated by alternative splicing at the C-terminus and previously thought to lack transactivation potential, are capable of activating wt1 expression. The endogenous wt1 promoter can be upregulated by exogenously supplied PAX8, suggesting that a function of PAX8 during mesenchymal--epithelial cell transition in renal development is to induce wt1 gene expression (Dehbi, 1996b).

The mammalian kidney develops in three successive steps from the initial pronephros via the mesonephros to the adult metanephros. The three successive steps are all characterized by the mesenchymal-to-epithelial transformation of intermediate mesoderm cells. The development of the first kidney, the transient pronephros, is initiated by signals from the somite and surface ectoderm that induce cells in the intermediate mesoderm to undergo the transition to epithelial cells forming the nephric duct. The caudal migration of the nephric duct subsequently induces the adjacent nephrogenic mesoderm to aggregate and form the tubules of the mesonephros, the second embryonic kidney. On further extension, the nephric duct reaches the metanephrogenic mesenchyme at the level of the developing hindlimb, where the ureteric bud evaginates from the nephric duct and invades the surrounding mesenchyme. Both the ureter and mesenchyme subsequently undergo reciprocal inductive interactions to form the nephrons and collecting ducts of the metanephros, the third and adult kidney. Ultimately, the development of the metanephros therefore depends on the proper formation of the nephric duct during pronephros induction. Although the nephric lineage is specified during pronephros induction, no single regulator, including the transcription factor Pax2 or Pax8, has yet been identified to control this initial phase of kidney development. Mouse embryos lacking both Pax2 and Pax8 are unable to form the pronephros or any later nephric structures. In these double-mutant embryos, the intermediate mesoderm does not undergo the mesenchymal-epithelial transitions required for nephric duct formation, fails to initiate the kidney-specific expression of Lim1 and c-Ret, and is lost by apoptosis 1 d after failed pronephric induction. Conversely, retroviral misexpression of Pax2 was sufficient to induce ectopic nephric structures in the intermediate mesoderm and genital ridge of chick embryos. Together, these data identify Pax2 and Pax8 as critical regulators that specify the nephric lineage (Bouchard, 2002).

Pax8 and thyroid development

Several mouse genes designated 'Pax genes' contain a highly conserved DNA sequence homologous to the paired box of Drosophila. Pax8 has an open reading frame of 457 amino acids (aa) contains the 128 aa paired domain near the amino terminus. Another conserved region present in some other paired box genes, the octapeptide Tyr-Ser-Ile-Asn-Gly-Leu-Leu-Gly, is located 43 aa C-terminal to the paired domain. Using an interspecies backcross system, the Pax8 gene was mapped within the proximal portion of mouse chromosome 2 in a close linkage to the surf locus. Pax8 is expressed during mouse embryogenesis transiently between 11.5 and 12.5 days of gestation along the rostrocaudal axis extending from the myelencephalon throughout the length of the neural tube, predominantly in two parallel regions on either side of the basal plate. Pax8 expression is also detected in the developing thyroid gland beginning at 10.5 days of gestation, during the thyroid evagination. In the mesonephros and metanephros the expression of Pax8 is localized to the mesenchymal condensations, which are induced by the nephric duct and ureter, respectively. These condensations develop to functional units, the nephrons, of the kidney. These data are consistent with a role for Pax8 in the induction of kidney epithelium. The embryonic expression pattern of Pax8 is compared with that of Pax2, another recently described paired box gene expressed in the developing excretory system (Plachov, 1990).

The murine and human Pax8 genes are expressed in developing and adult thyroid as well as in the developing secretory system and at the lower level in adult kidney. In the secretory system, expression is localized to the induced, extensively differentiating parts that undergo a transition from mesenchyme to epithelium. The human PAX8 gene generates at least five different alternatively spliced transcripts encoding different PAX8 isoforms. These isoforms differ in their carboxy-terminal regions downstream of the paired domain that is responsible for the DNA binding. The PAX8a isoform contains a 63 amino-acid serine-rich region that is absent in the isoform PAX8b, whereas PAX8c reveals a novel 99-amino-acid proline-rich region. This proline-rich region arises due to an unusual reading-frame shift in the PAX8 transcript. RNAse protection and RT(reverse transcription)-PCR analysis show the expression of all three PAX8 transcripts in human thyroid, kidney and five Wilms' tumors. Band-shift assay indicates a greatly reduced binding affinity of the isoform PAX8c to a DNA sequence from the promoter of the thyroperoxidase gene, as compared to the binding of PAX8a and PAX8b to this sequence. Deletion analysis of murine PAX8a indicates that its activating domain residues at the carboxy terminus of the protein which is shared by isoforms PAX8a and PAX8b. In accordance with these data PAX8a and PAX8b activate transcription from a thyroglobulin promoter as well as from a cotransfected synthetic PAX8-specific promoter. However, if the basal synthetic promoter of this reporter is substituted by a minimal TATA element, PAX8a and PAX8b fail to activate transcription. Only a GAL4-PAX8b fusion significantly activates transcription from a cotransfected GAL4-specific upstream-activating-sequence reporter. These results indicate that the PAX8 isoforms display different functional properties and may also function differently in vivo (Poleev, 1997).

Thyroid-enriched transcription factors, Pax-8 and TTF-1, are involved in the thyroid-specific expression of the thyroglobulin (TG) gene. Redox regulation of both factors has been shown to occur both in vitro and in vivo. When analyzed by electrophoretic mobility shift assay (EMSA), oxidation with diamide abolishes the DNA binding of Pax-8. Subsequent reduction with dithiothreitol (DTT) restores the binding. Thioredoxin (TRX), a cellular reducing catalyst, restores the binding more efficiently than DTT. When TTF-1 is oxidized with diamide, its binding is decreased and the TTF-1-DNA complex migrates faster on EMSA. DTT reverses these effects. These observations indicate that reduction is required for full DNA binding of Pax-8 and TTF-1 in vitro. TSH modulates their binding through redox regulation. Whole cell extracts were prepared from FRTL-5 cells at intervals after TSH treatment without reducing agents and subjected to EMSA. Pax-8 and TTF-1 binding activities are gradually increased during the initial 6 h after TSH. This increase is due to reduction of the factors, since treatment of the extracts with DTT mask the increase by enhancing their binding activities. These results suggest that TSH up-regulates the binding of Pax-8 and TTF-1, at least in part, by reducing the preexisting, oxidized forms in FRTL-5 cells. Northern analysis shows that the increase in TRX mRNA level by TSH in FRTL-5 cells is associated with the increase in the binding activities. Cotransfection of luciferase-reporter plasmid driven by TG promoter with Pax-8- and TRX-expressing plasmids into a heterologous cells reveals that TRX up-regulates the Pax8-mediated TG promoter activity. Taken together, the present study suggests that the redox regulation of Pax-8 and TTF-1 by TSH, probably through TRX, modulates the TG gene expression (Kambe, 1996).

back to Evolutionary homologs part 1/5 | part 2/5 |part 3/5 | part 4/5 |

sparkling: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.