BIOLOGICAL OVERVIEW

The paired ( prd) gene, once accorded a low significance rating in comparison with other pair-rule genes, has had its status elevated due to more recent information. Transcription of primary pair-rule genes is controlled directly by gap genes and maternal factors, while secondary pair-rule genes are considered to be regulated by the pair-rule primary genes (in addition to regulation by maternal and gap genes). paired is now thought to be a secondary pair rule gene. As such, it plays a decisive role in the progression of a regulatory hierarchy from pair-rule directed segmentation of the embryo to the subdivision carried out by segment polarity genes specifying positional information within segments.

The most recent analysis shows that early paired expression is determined by a combination of maternal factors and gap genes. The dissection of the paired promoter has revealed that all the other pair-rule genes are regulating the refinement of paired early stripe expression and the transition from an early 8 stripe pattern to a late 14 stripe pattern. A "prd zebra element" was found in the proximal promoter able to direct the expression of seven stripes; a 3' regulatory region regulates the anterior dorsal spot expression (Gutjahr 1993, 1994).

A specific function of paired is the development of the ventral chemosensory organ. Chemosensory organs are involved in gustatory and/or olfactory responses in larvae and/or adult flies. Part of the larval fly's feeding apparatus is a chemosensory structure known as the ventral organ, located near the mouth opening. This chemosensory organ consists of a pair of simple lobes with five small pores. These pores are innervated by the ventral ganglia that connect to the brain via the maxillary nerve.

An experimental manipulation demonstrates the involvement of paired in the development of this organ. Hammerhead ribozymes designed to cleave prd mRNA were placed under the regulation of heat shock promoters and induced during specific periods in the fly's larval development. Late stage embryos were exposed to heat for four to five hours, at a time when paired is expressed in the head. Treatment at this time avoids confusing possible effects with prd early stripe expression in head segments. In essence, the prd transcripts were knocked out by the ribozyme activity, drastically reducing Paired protein expression. The ventral chemosensory organs failed to develop, supporting the notion that paired is crucial for their normal development (Vanario-Alonso, 1995).

paired, whose product is homologous to the Drosophila Gooseberry and mammalian Pax3 proteins, has three general functions: proper development of the larval cuticle, survival to adulthood and male fertility. Both DNA-binding domains, the conserved N-terminal paired-domain (PD) and prd-type homeodomain (HD), are required within the same molecule for all general paired functions, whereas a conserved His-Pro repeat located near its C terminus is a transactivation domain potentiating these functions. The C-terminal moiety of Paired includes two additional functional motifs: one, also present in Gooseberry and Pax3, is required for segmentation and cuticle development; the other, retained only in Gooseberry, is necessary for survival. The male fertility function, which cannot be replaced by Gooseberry and Pax3, is specified by the conserved N-terminal rather than the divergent C-terminal moiety of Paired. It is concluded that the functional diversification of paired, gooseberry and Pax3, primarily determined by variations in their enhancers, is modified by adaptations of their coding regions as a necessary consequence of their newly acquired spatiotemporal expression (Xue, 2001).

With the aid of two alleles of prd, prd-Gsb and prd-Pax3, in which the gsb and Pax3 coding regions were placed under the control of the entire prd cis-regulatory region, it has been shown that Prd activity is required in vivo during at least three distinct developmental stages to ensure proper segmentation of the larval cuticle, postembryonic viability and male fertility. In this study, a series of prd transgenes were constructed that express various versions of the Prd protein, including truncations or chimeras of Prd, Gsb and Pax3 under the control of the complete prd cis-regulatory region. All transgenes were tested for their ability to rescue any of these Prd functions. Thus, this report is the first example of a complete functional analysis of the Prd protein under natural conditions (Xue, 2001).

The presence of two DNA-binding domains, PD and HD, in Prd and some other members of the Pax gene family raises the question of whether the regulation of any of its target genes requires the binding of both or only one of its two DNA-binding domains. Both mechanisms are compatible with in vitro results. In vivo studies show that both PD and HD are absolutely required for Prd function because deletion of either or both of these domains from the prd-Gsb transgene results in the complete loss of its ability to rescue the segment-polarity gene activation, cuticular phenotype and lethality of prd mutants. Moreover, since a point mutation in the PD (i.e., prd-Gsb^P17L) eliminates all Prd functions, the DNA-binding ability of the PD is necessary for the normal functions of Prd. An analogous mutation abolishes DNA binding of the human PAX5 protein and causes Waardenburg's syndrome I when present in PAX3 (Xue, 2001).

The observation that prd-GsbdeltaP and prd-GsbdeltaH cannot complement for any function of Prd implies that the PD and HD must be present in the same Prd molecule, presumably because each Prd function requires the recognition of at least one composite DNA target site. In agreement with these findings, Prd proteins unable to bind DNA as a result of single amino acid substitutions in either the PD or HD can no longer activate the ectopic expression of Prd-target genes when expressed ubiquitously under the control of the heat-shock promoter nor will these mutant proteins perform any Prd in vivo function when expressed under the control of some of prd enhancers. In addition, a composite Prd target site has been identified in the even-skipped enhancer whose mutation in either the PD or HD binding portion dramatically reduces Prd binding activity both in vitro and in vivo. The finding that the PD and HD cannot complement in trans for any function of Prd agrees with some observations obtained with mutant transgenes in vivo, but contradicts results obtained in vitro, and in vivo when the two Prd mutant proteins are expressed under heat-shock control. Taken together, these results imply that the PD and HD of Prd may interact with their DNA targets cooperatively and that this cooperativity can occur in trans only if the proteins are produced at concentrations much higher than those occurring naturally (Xue, 2001 and references therein).

The PRD repeat, which encodes a 20-30 amino acid His-Pro repeat, was discovered in an attempt to verify predictions of the gene network hypothesis in a search for protein-coding domains of prd. The PRD repeat is found in a number of Drosophila early developmental genes, including bicoid and daughterless, but its in vivo function remained unknown. Previous experiments in cell culture systems have shown that the PRD repeat is part of a transactivation domain that is necessary to drive ectopic expression of Prd-target genes under the control of ubiquitously expressed Prd. Other studies, however, have suggested that the PRD repeat is not essential for in vivo functions of Prd. The Prd protein whose PRD repeat has been deleted in prd-PrddeltaPRD is still able to perform all in vivo functions of Prd, which implies that the PRD repeat is not absolutely required for Prd function. However, the fact that one copy of prd-PrddeltaPRD exhibits significantly reduced efficiency in its ability to rescue the lethality and male sterility of prd mutants indicates that the PRD repeat greatly facilitates these Prd functions. This conclusion is corroborated and extended by the results obtained with prd-Gsb+PRD transgenes, which demonstrate that the PRD repeat enhances the viability as well as the cuticle function of Prd. Thus, the PRD repeat is an important transactivation domain that facilitates all functions of Prd (Xue, 2001).

Previous work has demonstrated that Prd, Gsb and Pax3 proteins are, at least partially, functionally equivalent. When expressed under the control of the entire cis-regulatory region of prd, both Gsb and Pax3 can activate Prd-target genes necessary for the generation of wild-type cuticle, while Gsb is able to rescue prd mutants to adulthood. These results strongly suggested that the acquisition of cis-regulatory regions rather than the divergence of their coding regions is the primary evolutionary mechanism responsible for the functional diversification of prd, gsb and Pax3 genes. However, although Gsb and Pax3 can substitute for most Prd functions, they do so at considerably reduced efficiencies: this indicates that these proteins had to adapt their new functions for optimal performance by subsequent mutations producing the observed divergence of the Prd, Gsb and Pax3 proteins. The result of this process of adaptation has been studied by examining the functional differences between these proteins when expressed as evolutionary alleles under the same cis-regulatory region (Xue, 2001).

The results lead to the idea that, in addition to the PRD repeat, two motifs or domains are present in the C-terminal portion of Prd, on whose functions the formation of wild-type larval cuticle and survival to adulthood depend. Although no significant similarity has been found among the primary sequences of the C-terminal moieties of Prd, Gsb and Pax3, the motif required for implementing wild-type cuticle is shared by all three proteins. In contrast, the motif necessary for Prd’s viability function is retained only in Gsb, presumably as secondary or tertiary protein structure. It should be stressed that at least two independent functions of Prd are required for viability, one of which, Pax3, is able to perform even better than Gsb. However, Pax3 is unable to substitute for one of the viablity functions of Prd and even exerts a dominant-negative effect on it. In agreement with this postulate, combining the results with those obtained with two weak prd alleles encoding truncated Prd proteins, allows the motifs for the cuticle and viability functions to be mapped within the C terminus of Prd (Xue, 2001).

Although prd-Gsb rescues prd mutants to viable adults, all males are sterile. Since wild-type males transgenic for two copies of prd-Gsb are fertile, it is concluded that prd has a function required for male fertility. Moreover, since prd-Gsb includes the entire cis-regulatory region of prd, its failure to rescue male fertility must be caused by the inability of Gsb to replace this function of the Prd protein. Since Prd and Gsb share a highly conserved N-terminal portion consisting of two DNA-binding domains, the PD and HD, it seemed plausible to map this functional difference to their divergent C termini. Surprisingly, however, the protein-domain-swapping experiments indicate that the conserved N-terminal rather than the divergent C-terminal portion is the determinant for this particular function of Prd. Therefore, it is suggested that at least one specific Prd target site, recognized by Prd but not Gsb, is involved in male fertility. The male fertility function of Prd is controlled by a specific prd enhancer uncovered in prd mutants by a prd rescue construct that lacks 5 kb of the downstream regulatory region. Consistent with this interpretation, a prd transgene that expresses Prd merely under the control of this 5 kb regulatory region is able to confer fertility to prd-Gsb males mutant for prd. Males completely deficient for this fertility function of prd have no accessory glands, while accessory glands begin to form in prd mutant males rescued by prd-Gsb, but stop development at a severely reduced size. These findings are in agreement with the hypothesis that new functions evolve primarily through the acquisition of new enhancers during gene duplication and that the adaptation of the protein is secondary and a necessary consequence of its expression in the newly acquired context of this function (Xue, 2001).

These results further imply that the C-terminal portions of Prd and Gsb, though divergent in their primary sequences, are still qualitatively the same. Hence, the validity of amino acid similarity as a general measure of functional equivalence in homologous proteins can be questioned. Instead, it has been proposed that this measure of functional equivalence should be replaced by calculations of the mutual entropy between two protein sequences, a more precise statistical measure that takes into account the probability by which certain amino acids are replaced by others (Xue, 2001 and references therein).

GENE STRUCTURE

Bases in 5' UTR - 264

Exons - two

Bases in 3' UTR - 350

PROTEIN STRUCTURE

Amino Acids - 613

Structural Domains

The Paired protein has a bipartite paired domain (PD), a central paired class homeodomain and a C-terminal PRD (His-Pro sequence) repeat (Frigerio, 1986). The Paired domain is a region of homology shared by certain homeodomain proteins called Paired homeodomains. The Paired repeat, is embedded in a proline rich transactivation domain (Cai, 1994).

Pax-3, the vertebrate Paired homolog, contains two structurally independent DNA-binding domains: a paired-domain and a homeodomain. Their functional interdependence has been suggested by the analysis of the Sp-delayed (Spd) mouse mutant, in which a glycine to arginine substitution at position 9 of the paired-domain abrogates DNA binding by both domains. This glycine is located in the beta-turn portion of a beta-hairpin motif; the requirement for this structure was investigated by mutagenesis at this and neighboring positions. At position 9, only substitution with proline increases DNA binding by the paired-domain and homeodomain above the level observed with the Spd arginine mutation, suggesting that the beta-turn is necessary for the function of both DNA-binding domains. Alanine scanning mutagenesis also identifies a number of flanking residues important for DNA binding by both domains, emphasizing the requirement of the beta-hairpin for the interaction of Pax-3 with DNA. These mutations reduce binding by the homeodomain at the monomeric level and do not impair dimerization on a TAAT(N)2ATTA consensus motif. In contrast, the wild-type paired-domain prevents dimerization on consensus motifs with 3-base pair spacing of the type TAAT(N)3ATTA. Importantly, both the deleterious effect of the Spd mutation on homeodomain DNA binding and the loss of dimerization on TAAT(N)3ATTA motifs can be transferred to a heterologous homeodomain from the human phox protein. Moreover, the presence of the paired-domain affects sequence discrimination within the 3-base pair spacer in this context. These analyses establish that the beta-hairpin motif is essential for paired-domain and homeodomain DNA binding, and suggest a novel mechanism by which the paired-domain can influence sequence specificity of the homeodomain within the Pax-3 polypeptide (Underhill, 1997).

date revised: 28 February 2001  

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