Promoter-enhancer interactions in two adjoining drosophila genes

9317701 Blackman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，我们将研究果蝇十肢麻痹（dpp）基因的基因内调控，因为我们试图解释其复杂的成虫盘特异性增强子网络如何寻找正确的启动子。 这些实验将验证我们的观点，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的调节自主性的性质。 尽管dpp增强子可以激活它们自己的启动子，最远可达60 kb，但它们对相邻的tnd（靠近dpp的转录本）基因的启动子是无关紧要的，而tnd基因的启动子仅位于几kb之外。 两个模型，一个涉及染色质结构域，另一个启动子特异性，通常被用来描述基因如何保持自主性，我们处于一个很好的位置，以确定这些，如果其中之一，是在dpp/tnd系统中工作。 通过我们提出的实验：1）我们将检查不同盘特异性增强子的启动子偏好，并确定这些选择是否通过启动子竞争机制建立。 这项工作的大部分将涉及组织培养细胞中的转染测定，但这些结果也将通过体内和分析来证实。 2)我们将表征TND转录所需的调节序列。 我们将使用报告基因构建体和种间序列比较来鉴定组成tnd启动子和增强子区域的功能重要元件。 3)我们将建立一个机制，让国民党和民进党保持自主性。 我们将使用“启动子交换”实验来决定是否调控独立性是通过染色质结构域或启动子特异性介导的。 对于这些实验，我们将使用体内定点诱变来用另一个完全替换tnd启动子。 4)我们将精确地确定哪些染色体序列负责这种调节分离。 我们将使用分子、分子遗传学和生物化学技术来鉴定所涉及的元素。 基因在真核系统中是如何被调控的还不清楚。被称为增强子的序列与特定的启动子序列相互作用以诱导由启动子控制的基因的表达，但是由于增强子可以在一定距离处并且在任一方向上，因此特定序列如何相互作用尚未被理解。由于该系统的新功能，再加上强大的分子遗传技术可用于果蝇，这项工作的结果应该提供一个最清晰的理解增强子如何区分替代启动子。 ***

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. ***