Crossgrove K, Bayer C A, Fristrom J W, Guild G M
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6018, USA.
Dev Biol. 1996 Dec 15;180(2):745-58. doi: 10.1006/dbio.1996.0343.
The ensemble of tissue-specific changes that drives Drosophila metamorphosis is initiated by the steroid hormone ecdysone and proceeds through a transcriptional cascade comprised of primary response transcriptional regulators and secondary response structural genes. The Broad-Complex (BR-C) primary response early gene is composed of several distinct genetic functions and encodes a family of related transcription factor isoforms. Our objective in this study was to determine whether individual BR-C isoforms directly regulate secondary response target genes. A cluster of 10 salivary gland-specific secondary response L71 late genes are dependent on the BR-C rbp+ genetic function. Transgenic animals expressing individual BR-C isoforms were tested for their ability to provide the BR-C rbp+ genetic function by monitoring the transcriptional activation of the L71 genes. We found that the BR-C Z1 isoforms could complement the transcriptional defects seen in rbp mutants but the Z2, Z3, and Z4 isoforms could not. We conclude that the BR-C rbp+ function is provided by the BR-C Z1 isoform in prepupal salivary glands. L71 gene rescue was restricted to the prepupal salivary gland, suggesting the involvement of additional factors in L71 gene regulation. Interestingly, we found that the overexpression of Z3 or Z4 isoforms in BR-C+ salivary glands repressed L71 expression, indicating that BR-C proteins might also function as transcriptional repressors. Molecular mapping and characterization of the regulatory sequences that control L71-6 expression revealed several Z1 isoform binding sites. Mutagenesis of these Z1 binding sites resulted in the failure to activate late gene expression in vivo when measured by transgenic reporter genes. We conclude that the BR-C early gene directly activates late gene transcription by interacting with late gene cis-acting regulatory elements and that this interaction is responsible for the temporal linkage of early and late ecdysone-induced gene expression.
驱动果蝇变态的组织特异性变化整体由类固醇激素蜕皮激素启动，并通过一个由初级反应转录调节因子和次级反应结构基因组成的转录级联反应进行。泛素连接酶复合体（BR-C）初级反应早期基因由几种不同的遗传功能组成，并编码一系列相关的转录因子异构体。我们在本研究中的目的是确定单个BR-C异构体是否直接调节次级反应靶基因。一组10个唾液腺特异性次级反应L71晚期基因依赖于BR-C rbp+遗传功能。通过监测L71基因的转录激活，测试了表达单个BR-C异构体的转基因动物提供BR-C rbp+遗传功能的能力。我们发现BR-C Z1异构体可以弥补rbp突变体中出现的转录缺陷，但Z2、Z3和Z4异构体则不能。我们得出结论，在蛹前期唾液腺中，BR-C rbp+功能由BR-C Z1异构体提供。L71基因拯救仅限于蛹前期唾液腺，这表明在L71基因调控中还涉及其他因素。有趣的是，我们发现BR-C+唾液腺中Z3或Z4异构体的过表达会抑制L71表达，这表明BR-C蛋白也可能作为转录抑制因子发挥作用。对控制L71-6表达的调控序列进行分子定位和表征，发现了几个Z1异构体结合位点。通过转基因报告基因测量，这些Z1结合位点的诱变导致体内晚期基因表达无法激活。我们得出结论，BR-C早期基因通过与晚期基因顺式作用调控元件相互作用直接激活晚期基因转录，并且这种相互作用负责早期和晚期蜕皮激素诱导基因表达的时间联系。
