Mouse Hox-2.2 specifies thoracic segmental identity in Drosophila embryos and larvae

The mouse genome has a number of homeobox genes that are structurally similar to the Drosophila Antenapedia (Antp) gene. We find that one of the mouse Antp-like genes, Hox-2.2, when expressed in developing Drosophila cells under control of a heat shock promoter, can induce homeotic transformations that are nearly identical to those caused by ectopic expression of Antp. In larvae, the Hox-2.2-induced transformations include thoracic denticle belts in place of head structures; in adults, the Hox-2.2 transformations include thoracic legs in place of antennae. The phenotypic effects of Hox-2.2 do not depend on the endogenous Antp gene, whose spatial limits of expression are unaffected by Hox-2.2 expression. Thus, in the Drosophila embryo, Hox-2.2 can substitute for some of the segmental identity functions of Antp, presumably by regulating the same set of downstream genes.     

Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.

Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities.     

Structure and expression of ubiquitin genes of Drosophila melanogaster.

We isolated and characterized two related ubiquitin genes from Drosophila melanogaster, polyubiquitin and UB3-D. The polyubiquitin gene contained 18 repeats of the 228-base-pair monomeric ubiquitin-encoding unit arranged in tandem. This gene was localized to a minor heat shock puff site, 63F, and it encoded a constitutively expressed 4.4-kilobase polyubiquitin-encoding mRNA, whose level was induced threefold by heat shock. To investigate the pattern of expression of the polyubiquitin gene in developing animals, a polyubiquitin-lacZ fusion gene was introduced into the Drosophila genome by germ line transformation. The fusion gene was expressed at high levels in a tissue-general manner at all life stages assayed. The ubiquitin-encoding gene, UB3-D, consisted of one ubiquitin-encoding unit directly fused, in frame, to a nonhomologous tail sequence. The amino acid sequence of the tail portion of the protein had 65% positional identity with that of yeast UBI3 protein, including a region that contained a potential nucleic acid-binding motif. The Drosophila UB3-D gene hybridized to a 0.9-kilobase mRNA that was constitutively expressed, and in contrast to the polyubiquitin gene, it was not inducible by heat shock.     

Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.     

Regulation of proboscipedia in Drosophila by homeotic selector genes

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. We have identified several of the genes necessary to generate this embryonic pattern of expression. These genes can be roughly split into three categories based on their time of action during development. First, prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. Second, the initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). Lastly, maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. We further show that Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila melanogaster and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct.