THE MECHANISMS BY WHICH HELIX-LOOP-HELIX TRANSCRIPTION

为了了解决定神经细胞命运的分子机制，我们正在研究毛发相关的碱基螺旋-环-螺旋(BHLH)抑制蛋白在神经发育中的作用机制。我们先前已经证明，毛状bHLH蛋白确实与无毛类原神经基因中的单个CACGCG位点结合，抑制无毛类转录，从而阻止果蝇异位感觉器官的形成。在这项研究中，我们研究了毛发蛋白是如何通过与靶基因结合来抑制转录的。通过瞬时转染实验，我们已经证明了毛发相关的bHLH抑制物的羧基末端的WRPW基序作为4个氨基酸的转录抑制域和与Grucho相关的辅阻遏物的蛋白质相互作用结构域。我们还发现，当Gruocho相关蛋白直接与靶基因启动子结合时，可以主动抑制转录，并在这些蛋白中发现了一个新的、高度保守的转录抑制域。此外，利用转基因果蝇，我们已经清楚地表明，毛发相关抑制子的WRPW基序和Groucho相关辅阻遏子的氨基末端转录抑制域可以通过异源蛋白Gal4靶向achaete基因而发挥抑制果蝇感觉器官形成的作用。这些结果有力地支持了一种模型，即与远端CACGCG位点结合的毛发bHLH蛋白通过WRPW基序将Groucho辅阻遏物蛋白招募到achaete启动子，从而主动抑制achaete转录，以防止果蝇感觉器官在错误位置形成。

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.