Genetic evidence for the transcriptional-activating function of Homothoraxduring adult fly development

Homothorax (HTH) is a homeobox-containing protein, which plays multiple rol es in the development of the embryo and the adult fly. HTH binds to the hom eotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-con taining complexes in the context of the developing fly. Here we present gen etic evidence, from in vivo studies, for the transcriptional-activating fun ction of HTH. The HTH protein was forced to act as a transcriptional repres sor by fusing it to the Engrailed (EN) repression domain, or as a transcrip tional activator, by fusing it to the VP16 activation domain, without pertu rbing its ability to translocate EXD to the nucleus. Expression of the repr essing form of HTH in otherwise wild-type imaginal discs phenocopied hth lo ss of function. Thus, the repressing form was working as an antimorph, sugg esting that normally HTH is required to activate the transcription of downs tream target genes. This conclusion was further supported by the observatio n that the activating form of HTH caused typical hth gain-of-function pheno types and could rescue hth loss-of-function phenotypes. Similar results wer e obtained with XMeis3, the Xenopus homologue of HTH, extending the known f unctional similarity between the two proteins. Competition experiments demo nstrated that the repressing forms of HTH or XMeis3 worked as true antimorp hs competing with the transcriptional activity of the native form of HTH. W e also describe the phenotypic consequences of HTH antimorph activity in de rivatives of the wing, labial and genital discs. Some of the described phen otypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH prot ein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.