In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product

Abstract

The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the N- terminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.

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

“…These observations lead to two predictions. First, the C‐terminal region of the paired domain appears to make only a minor contribution to the DNA binding of Pax3 and Pax7 in analogy to the corresponding Drosophila homologue Paired (Cai et al ., 1994; Bertuccioli et al ., 1996; Jun and Desplan, 1996). Second, the DNA‐binding specificity of Pax proteins can be modified through alternative splicing in two different ways by inserting extra amino acids either into the N–terminal (Pax6 and Pax8) or C‐terminal (Pax3 and Pax7) region of the paired domain.…”

“…X‐ray crystallographic analysis of the paired domain–DNA complex recently confirmed this bipartite model by demonstrating that each of the two subdomains is composed of a helix–turn–helix (HTH) motif resembling the structure of the homeodomain (Xu et al ., 1995). Interestingly, the paired domain of the Drosophila Paired protein relies entirely on the N‐terminal subdomain for DNA binding (Jun and Desplan, 1996) in agreement with the observation that the C‐terminal motif is dispensable for normal paired function in vivo (Cai et al ., 1994; Bertuccioli et al ., 1996). In contrast, the paired domain of Pax6 depends on both subdomains for DNA sequence recognition similar to all members of the Pax2/5/8 family (Epstein et al ., 1994b; Czerny and Busslinger, 1995).…”