Functional specificity of the Antennapedia homeodomain ( Sex comb reduced / heat shock / segmental identity / gene expression / homeotic btandormation

The segmental identity in animal development is determined by a set ofhomeotic selector genes clustered in the invertebrate HOM or vertebrate Hox homeo box complexes. These genes encode proteins with very smilar homeodomains and highly diverged Nand C-terminal sequences. The Antennapedia (Antp) homeodomain, for Instance, differs at only five amino acid positions from that of Sex combs reduced (Scr) protein. Using a heat shock assay in which chimeric Antp-Scr proteins are expressed ectopicafly in Drosophila, we have shown that the functional specdflcity of the Antp protein is determined by the four specfic amino acids located in the flexible N-terminal arm ofthe homeodomain. The three-dimensional structure of the Antp homeodomain-DNA comnplex shows that this N-terminal arm is located in the minor groove of the DNA, suggesting that the functional specificity is determined either by slight differences in DNA binding and/or by selective interactions with other transcription factor(s). The body ofthe fruitfly is composed ofa series ofconsecutive segments arranged along the anteroposterior axis, each of which shows a distinct identity. The genetic and molecular studies of Drosophila development have identified a set of homeotic selector genes that specify the morphological and functional identity of each of these metameric segments (reviewed in ref. 1). The homeotic genes are clustered in the Antennapedia Complex (ANTC) and Bithorax Complex (BXC) on the third chromosome encoding nuclear transcription factors that contain the homeodomain, a 60-amino acid DNA-binding domain. The protein sequences encoded by the members of the ANTC and BXC genes are largely different from each other except for their homeodomains, which show strong amino acid sequence conservation including almost identical recognition helices (2). Previous studies have indicated that the functional specificity of these homeotic selector proteins is determined by the C-terminal parts of the proteins that include their homeodomains (3-5). In this paper, we show that the four amino acid residues at positions 1, 4, 6, and 7 in the flexible N-terminal arm of the homeodomain determine the functional specificity of the Antp protein in transgenic flies. In the three-dimensional structures ofthe homeodomain-DNA complexes (6-8), this N-terminal arm is located in the minor groove ofDNA. Our results suggest that the functional specificity of the homeotic selector proteins is determined either by a slight difference inDNA binding and/or by selective association with other specific transcription factors. MATERIALS AND METHODS Gene Construction. The four amino acid changes were introduced into the plasmids pHSS-AfSL and pHSS-SA26 (5) by substituting the Bsm I-Xmn I fragments with the doublestranded oligonucleotide AAT GGC GAG CGC AAA CGC The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. GGAAGG CAG ACA TAC ACC CGG TAC CAG ACT CTA GAG CTA GAG AAG G. The mutated gene was subsequently subcloned into the unique Not I site of pNHT4 (5). During the confirmation of the mutant sequence, we found that six residues are missing from the reported Scr (Sex combs reduced) sequence 1460-1600 (9). The corrections have been submitted to the EMBO data base. P-Element Transformation and Maintenamce ofFlies. Transgenic flies were generated by P-element transformation and maintained as described (5). The rosy eye ry6 allele was used as recipient of the transposon. Only two or three lines were recovered from 1000 injections probably because of the toxicity of the encoded proteins expressed by leaky transcription of the heat shock gene Hsp7O promoter. Heat Shocks. Embryonic and larval heat shocks were applied as described (5). Briefly, embryos were heat-shocked for 20 min at 37°C 6-7 hr after egg laying and were allowed to develop at 25°C for 36 hr. For larvae, six consecutive heat shocks at 37°C for 60 min were applied at 4-hr intervals from 69 ± 0.5 hr to 88 ± 0.5 hr after egg laying.