Promoter-enhancer interactions in two adjoining drosophila genes

9317701布莱克曼：我们的实验将探索两种涉及启动子-增强子相互作用的基因调控模式。首先，我们将研究果蝇十五麻痹(DPP)基因的基因内调控，试图解释其复杂的盘特异性增强子网络是如何寻找到正确的启动子的。这些实验将检验我们的观点，即这是通过启动子竞争机制发生的。我们的第二条实验路线将调查相邻基因调节自主性的本质。尽管DPP增强子可以激活60kb以外的自己的启动子，但它们对邻近TND(Transcript Near DPP)基因的启动子无动于衷，而TND基因位于几kb之外。两个模型，一个涉及染色质结构域，另一个涉及启动子特异性，通常被用来描述基因如何保持自主，我们处于有利的地位，以确定这两种模型中的哪一种，如果有的话，在DPP/TND系统中发挥作用。通过我们提出的实验：1)我们将检查不同的盘特异性增强子的启动子偏好，并确定这些选择是否是通过启动子竞争机制建立的。这项工作的大部分将涉及组织培养细胞的转基因分析，但这些结果也将得到体内和分析的证实。2)我们将确定TND转录所需的调控序列。我们将使用报告基因构建和物种间序列比较来确定组成TND启动子和增强子区域的重要功能元件。3)建立民进党和民进党自治机制。我们将使用“启动子交换”实验来确定调控独立性是通过染色质结构域还是启动子专一性来介导的。对于这些实验，我们将使用体内定点突变来完全用另一个启动子取代TND启动子。4)我们将准确地确定是什么染色体序列负责这种调节分离。我们将使用分子、分子遗传和生化技术来鉴定所涉及的元素。%基因在真核系统中是如何调控的，目前还不清楚。被称为增强子的序列与特定的启动子序列相互作用，诱导由启动子控制的基因的表达，但由于增强子可以在一定距离和方向上，特定序列如何相互作用尚不清楚。由于这个系统的新特征，再加上果蝇可用的强大的分子遗传学技术，这项工作的结果应该会为增强剂如何区分不同的启动子提供最清晰的理解之一。***

9317701 Blackman Our experiments will explore two modes of gene regulation involving promoter-enhancer interactions. In the first, we will look at the intragenic regulation of the Drosophila decapentaplegic (dpp) gene, as we try to explain how its complex network of imaginal disk- specific enhancers search out their correct promoters. These experiments will test our view that this occurs through a mechanism of promoter competition. Our second line of experimentation will investigate the nature of the regulatory autonomy of adjacent genes. Even though the dpp enhancers can activate their own promoters up to 60 kb away, they are indifferent to the promoter of the adjacent tnd (transcript near dpp) gene which is located just a few kb away. Two models, one involving chromatin domains and the other promoter specificity, are commonly invoked to describe how genes can remain autonomous and we are in an excellent position to decide which of these, if either, is at work in the dpp/tnd system. Through our proposed experiments: 1) we will examine the promoter preferences of different disk-specific enhancers and determine whether these choices are established through a mechanism of promoter competition. Much of this work will involve transfection assays in tissue culture cells but these results will also be confirmed by in vivo and analysis. 2) we will characterize the regulatory sequences required for tnd transcription. We will use reporter gene constructs and interspecies sequence comparisons to identify the functionally important elements comprising the promoter and enhancer regions of tnd. 3) we will establish the mechanism by which tnd and dpp remain autonomous. We will use "promoter swap" experiments to decide whether the regulatory independence is mediated via chromatin domains or promoter specificity. For these experiments, we will use in vivo site- directed mutagenesis to completely replace the tnd promoter with another. 4) we will determine precisely w hich chromosomal sequences are responsible for this regulatory isolation. We will use molecular, molecular genetic, and biochemical techniques to identify the elements involved. %%% How genes are regulated in eukaryotic systems is not yet clearly understood. Sequences known as enhancers interact with specific promoter sequences to induce the expression of the gene controlled by the promoter but since the enhancer can be at a distance and in either orientation, how the specific sequences interact is not yet understood. Because of the novel features of this system, coupled with the powerful molecular genetic techniques available for Drosophila, the results of this work should provide one of the clearest understandings of how enhancers discriminate between alternative promoters. ***