THE MECHANISMS BY WHICH HELIX-LOOP-HELIX TRANSCRIPTION

为了了解神经细胞命运决定的分子机制，我们正在研究Hairy-related basec helix-loop-helix（bHLH）阻遏蛋白在神经发育中的作用机制。我们以前已经表明，毛茸茸的bHLH蛋白结合到一个单一的CACGCG网站在achaete proneural基因，抑制achaete转录，并作为一个结果块异位感觉器官的形成在果蝇。在这项研究中，我们解决了如何毛茸茸的bHLH蛋白抑制转录结合到一个靶基因。使用瞬时转染试验，我们已经证明，羧基末端WRPW基序的头发相关的bHLH阻遏物作为一个4-氨基酸的转录抑制结构域和蛋白质相互作用结构域与格鲁乔相关的辅阻遏物。我们还表明，Gruocho相关蛋白直接结合到靶基因启动子时，积极抑制转录，并确定了一个新的，高度保守的转录抑制结构域在这些蛋白质。此外，使用转基因苍蝇，我们已经清楚地表明，WRPW基序的头发相关的阻遏物和氨基末端的转录阻遏结构域的Groucho相关的辅阻遏物可以作为果蝇感觉器官形成的阻遏物通过靶向achaete基因通过异源蛋白Gal 4。这些结果有力地支持了一个模型，即毛bHLH蛋白结合到远端CACGCG网站积极抑制achaete转录的Groucho辅阻遏蛋白的招聘通过WRPW基序的achaete启动子，以防止感觉器官的形成在果蝇中的不正确的位置。

To understand the molecular mechanisms of neural cell fate determination, we are studying on the mechanism of action of the Hairy-related basec helix-loop-helix (bHLH) repressor proteins in neural development. We have previously shown that the Hairy bHLH protein does bind to a single CACGCG site in the achaete proneural gene, represses achaete transcription and as a consequence blocks ectopic sensory organ formation in Drosophila. In this study, we addressed how the Hairy bHLH protein represses transcription by binding to a target gene. Using transient transfection assays, we have demonstrated that the carboxyo-terminal WRPW motif of the Hairy-related bHLH repressors acts as a 4-amino-acid transcription repression domain and a protein interaction domain with the Grucho-related corepressors. We have also shown that the Gruocho-related proteins actively repress transcription when directly bound to a target gene promoter and identified a novel, highly conserved transcriptional repression domain in these proteins. Moreover, using transgenic flies, we have clearly shown that the WRPW motif of the Hairy-related repressors and the amino-terminal transcriptional repression domains of the Groucho-related corepressors can function as repressors of Drosophila sensory organ formation by targeting to the achaete gene through a heterologous protein Gal4. These results strongly support a model that the Hairy bHLH protein bound to the distally located CACGCG site actively represses achaete transcription by recruitment of the Groucho corepressor protein via the WRPW motif to the achaete promoter to prevent sensory organ formation in the incorrect locations in Drosophila.