To investigate the evolutionary conservation of proteins related to the GAGA factor of Drosophila melanogaster, a cDNA expression library from honeybees (A. mellifera) was screened using a radiolabeled probe containing GAGA factor binding sites. The probe was obtained by concatemerization of an oligonucleotide, hspGAGA1, that has been used to isolate GAGA factor cDNAs by expression screening. From 4.3 × 106 cDNA clones screened, three clones reproducibly showed strong binding of the GAGA probe. Southern hybridization and sequencing revealed that only one of these clones represented a cDNA from A. mellifera. The cDNA, referred to as AmPsq, contains an open reading frame encoding a 652-amino acid protein that shows high similarity to the Psq protein of D. melanogaster. Although overall sequence conservation is only 33%, two domains show strikingly high conservation. (1) The BTB/POZ domain is 73% identical with the BTB/POZ domain of D. melanogaster Psq. This means that despite the large evolutionary distance between D. melanogaster and A. mellifera, the degree of similarity between these domains is higher than the average identity of 53% between the BTB/POZ domains of different Drosophila proteins. (2) The region containing the four Psq repeats shows 80% identity with the Psq domain of the Drosophila protein. At the same time, not only the amino acid sequence of the single repeat units, but also the sequence of the repeats themselves is conserved. It is therefore concluded that AmPsq encodes the Psq homolog of A. mellifera. The sequence between the BTB/POZ and Psq domains of D. melanogaster Psq contains several regions that are particularly rich in certain amino acid residues, including two glutamine-rich regions and a region of 17 histidine residues alternating with other residues. Regions of this kind are believed to serve as interfaces for protein-protein interactions and are frequently found in transcription factors. Polyglutamine tracts, in particular, have the potential to interact with components of the basal transcription machinery and can thus act as transcription activation domains. The interdomain sequence of the A. mellifera protein, which is only half as long as the interdomain sequence of Drosophila Psq, lacks these regions of special amino acid composition and contains a single asparagine-rich region instead. Since such asparagine-rich regions are also present in many transcription factors, particularly homeodomain proteins, it is speculated that, despite their low sequence identity of only 13%, the interdomain regions of A. mellifera and D. melanogaster Psq have similar functions in both proteins (Lehmann, 1998).
To test the hypothesis that the AmPsq Psq domain might represent a novel DNA-binding domain, plasmids encoding truncated Psq proteins, as well as full-length Psq, were constructed and expressed using a reticulocyte in vitro translation system. Psq proteins were correctly expressed in the in vitro system. However, using a mobility shift DNA binding assay, no binding to the hspGAGA1 oligonucleotide was detected -- neither of full-length Psq nor of the truncated derivatives. Considering the repetitive structure of the Psq domain and the fact that a concatenated probe had been used for library screening, it is suspected that a more extended binding sequence might be required for Psq binding. Binding of the Psq proteins to oligonucleotide hspGAGA2, which is equivalent to the ligation product of two hspGAGA1 oligonucleotides, was tested. While binding of full-length Psq could not be detected also with this probe, a truncated form of Psq that lacks an essential part of the BTB/POZ domain shows strong binding (Psq Delta240). This result is consistent with the finding of Bardwell (1994) that BTB/POZ domains generally inhibit the interaction of their associated DNA-binding domains with DNA. If the N-terminal truncation of Psq is further extended to essentially remove the N-terminal half of the protein (Psq Delta333), the DNA binding ability is retained. Even if the truncation advances into the first amino acid residues of the Psq domain (removing the first 15 amino acids of Psq repeat 1), the resulting 268 amino acid protein (Psq Delta384) is still able to recognize the DNA target sequence. However, if the truncation removes the first two Psq repeats (Psq Delta474), DNA binding ability is lost. Truncations removing the Psq domain but leaving the BTB/POZ domain intact (Psq Delta232 and Psq Delta282) result in proteins that exert no specific DNA binding. Since full-length Psq also does not appear to bind to DNA unless the BTB/POZ domain is removed, this result is not surprising. However, it is interesting to note that complexes formed by proteins that are probably translated from internal AUG start sites are absent in lanes loaded with the Psq Delta232 and Psq Delta282 translation products. Such complexes are observed in all lanes loaded with translation products of constructs encoding a Psq domain, whether they encode a BTB/POZ domain or not. This suggests that products lacking both the Psq and BTB/POZ domain are also unable to bind DNA. Taken together, these data indicate that the Psq domain is responsible for DNA binding of the Psq protein. Since Psq repeats 3 and 4 together (Psq Delta474) are not sufficient for DNA binding, it seems likely that at least three complete repeat units are required for DNA binding. This is consistent with the requirement for a comparatively long DNA target sequence to detect binding. Interestingly, Horowitz (1996) isolated a cDNA encoding a putative Drosophila Psq isoform that resembles the Psq Delta384 derivative in that it also lacks the first 15 amino acid residues of Psq repeat 1. The deletions in both the D. melanogaster isoform and Psq Delta384 leave Psq repeats 2-4 as well as the putative helix-turn-helix motif of Psq repeat 1 intact. Since Psq Delta384 is still able to bind DNA, albeit with reduced affinity, this is probably also true for the Drosophila isoform. It is intriguing to speculate that a reduced binding affinity or altered specificity of this isoform directs it to a specific subset of Psq binding sites in vivo (Lehmann, 1998).
It was next asked whether binding of the Psq domain to DNA depends on the nucleotide sequence of the DNA or if it recognizes DNA in a rather nonspecific manner. Binding of Psq Delta240 to the radiolabeled hspGAGA2 oligonucleotide is specifically inhibited already at a 10-fold molar excess of the nonlabeled oligonucleotide. Two different oligonucleotides, similar in length and G/C content to hspGAGA2 but of different nucleotide sequence, show no or only slight competition even at a 100-fold molar excess over the probe. These data show that Psq binds to GAGA sequences with high sequence specificity (Lehmann, 1998).
Binding of Psq to DNA is greatly stimulated by the addition of magnesium to the incubation medium. Interestingly, this effect is observed with all DNA-binding Psq derivatives other than Psq Delta384, which lacks the N-terminal 15 amino acid residues of Psq repeat 1. One possible explanation for this finding is that the deletion in Psq Delta384 removes a Mg2+ binding site, possibly located at the N terminus of the Psq domain, that is essential for high affinity binding. It is important to note that the deletion in Psq Delta384 does not alter the binding specificity of the Psq domain, since binding of Psq Delta384 shows the same sensitivity toward different competitor DNAs as binding of Psq Delta240 (Lehmann, 1998).
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