Inbal A, Halachmi N, Dibner C, Frank D, Salzberg A
Unit of Genetics, Department of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Development. 2001 Sep;128(18):3405-13. doi: 10.1242/dev.128.18.3405.
Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic 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-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, 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 protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.
同胸蛋白（HTH）是一种含同源异型框的蛋白质，在胚胎和成年果蝇的发育过程中发挥多种作用。HTH与同源异型辅因子额外齿状蛋白（EXD）结合，并将其转运至细胞核。其在细胞核内的功能尚不清楚。主要通过体外研究表明，HTH可作为三元HTH/EXD/HOX复合物的一部分与DNA结合，但在发育中的果蝇体内，对含HTH复合物的转录调节功能了解甚少。在此，我们通过体内研究提供了关于HTH转录激活功能的遗传学证据。通过将HTH蛋白与 engrailed（EN）抑制结构域融合，使其被迫作为转录抑制因子发挥作用，或者通过将其与VP16激活结构域融合，使其作为转录激活因子发挥作用，同时不干扰其将EXD转运至细胞核的能力。在其他方面为野生型的成虫盘盘中表达抑制形式的HTH，模拟了hth功能丧失的表型。因此，抑制形式起到了反形态的作用，表明正常情况下HTH是激活下游靶基因转录所必需的。这一结论进一步得到以下观察结果的支持：激活形式的HTH导致典型的hth功能获得性表型，并且可以挽救hth功能丧失性表型。用HTH的非洲爪蟾同源物XMeis3也得到了类似的结果，扩展了这两种蛋白质之间已知的功能相似性。竞争实验表明，HTH或XMeis3的抑制形式作为真正的反形态与天然形式的HTH的转录活性相互竞争。我们还描述了HTH反形态活性在翅、唇和生殖盘衍生物中的表型后果。一些所描述的表型，例如喙到腿的转变，以前与HTH活性的改变无关。观察HTH反形态干扰不同发育途径的能力可能会引导我们找到HTH的新靶点。本文所述的HTH反形态提供了一种新方法，通过这种方法可以在任何所需的细胞或组织中以可诱导的方式阻断内源性HTH蛋白的转录活性，而不干扰EXD的核定位。
