Dissecting the functions of a novel cis element Promoter Targeting Sequence

描述(由申请人提供)：我们从Drosophiht Bithorax复合体中鉴定出一种新的顺式调控元件，启动子靶向序列(PTS)。PTS具有抗绝缘体活性，允许原本被阻止的增强子在介入性绝缘体上激活其启动子；它还具有启动子靶向活性，选择性地仅靶向两个可能的启动子中的一个。PTS位于腹型B(Abd-B)基因中，其调节区由Fab-7和Fab-8等绝缘体DNA组织成节段特异性结构域。PTS的突变导致ABD-B表达的丧失和腹部的同源转化。我们认为，PTS通过克服Fab绝缘子的增强子封闭活性，从而将Fab绝缘子转化为局部结构域边界元素来介导增强子-启动子之间的相互作用。我们的工作假设是，PTS通过在增强子周围的DNA和启动子附近的DNA之间形成稳定的结合来发挥作用，这种机制对绝缘子阻断不敏感，表观遗传稳定，并且不依赖于与增强子相互作用的激活剂。鉴于果蝇和脊椎动物在HOX基因簇中的惊人相似性，我们有理由相信PTS代表了一类新的顺式调控元件，在无脊椎动物和脊椎动物中调控HOX基因簇中的长程增强子-启动子相互作用。我们建议通过检验与我们的“稳定联想模型”相关的几个假设来剖析PTS的功能。在特定的目标中，我们将测试可遗传的稳定的增强子启动子相互作用的形成不依赖于增强子的身份和增强子结合蛋白的预测。在lb中，我们将测试PTS是否使绝缘体失活，以及绝缘体是否有助于PTS的启动子靶向活性。最后，在AIM LC中，我们将确定PTS相对于增强子、启动子和绝缘体的相对位置如何影响其抗绝缘子和启动子靶向活性，以及PTS如何影响ABD-B中增强子-启动子的特异性。在第二个特定目标中，我们将确定是否有任何已知的HOX基因调节因子介导或拮抗PTS活性。我们还将研究染色质修饰在PTS功能中的作用。最后，我们将进行基因筛选，以分离通过PTS元件发挥功能的基因或蛋白质。我们将主要关注一个全面的F1 FIP-FRT筛查，以确定显性和隐性突变。改变PTS活性的突变将被绘制成图谱，以确定修饰基因编码的蛋白质。鉴定介导PTS活性的蛋白质对于确定这些活性的分子机制是至关重要的，并将指导未来旨在了解如何在复杂的发育基因中调节增强子-启动子相互作用的研究。

DESCRIPTION (provided by applicant): We have identified a novel cis-regulatory element, the Promoter Targeting Sequence (PTS) from the Drosophiht Bithorax Complex. The PTS has an anti-insulator activity, allowing an otherwise blocked enhancer to activate its promoter over an intervening insulator; it also has a promoter-targeting activity, selectively targeting only one out of wo possible promoters. The PTS is located in the Abdominal-B(Abd-B) gene where the regulatory region is organized into segmental-specific domains by insulator DNAs such as Fab-7 and Fab-8. Mutations in the PTS result in the loss of Abd-B expression and homeotic transformations in the abdomen. We propose that PTS mediates enhancer-promoter interactions by overcoming the enhancer-blocking activity of the Fab insulators, thus converting the Fab insulators into local domain boundary elements. Our working hypothesis is that PTS functions by forming a stable association between DNA around the enhancer and the DNA near the promoter through a mechanism that is insensitive to insulator block, epigenetically stable, and independent of activators that interact with the enhancer. Given the striking similarity in Hox clusters between Drosophila and vertebrate, we have reason to believe that PTS represent a new class of cis-regulatory elements that regulate long-range enhancer-promoter interactions in the Hox gene clusters in both invertebrate and vertebrate animals. We propose to dissect the function of the PTS by testing several hypothesis related to our "stable association model". In Specific Aim la we will test the prediction that formation of a heritable stable enhancer promoter interaction is independent of enhancer identity and enhancer-binding proteins. In lb we will test whether or not the PTS inactivates an insulator, and whether or not the insulator contributes to the promoter-targeting activity of the PTS. Finally, in Aim lc, we will determine how the relative position of the PTS to an enhancer, promoter and insulator affects its anti-insulator and promoter-targeting activities, and how the PTS affects enhancer-promoter specificity in Abd-B. In the second specific aim, we will determine if any of the known Hox gene regulators mediate or antagonize PTS activities. We will also investigate the role of chromatin modification in PTS function. And lastly, we will conduct genetic screens to isolate genes or proteins that function through the PTS element. We will mainly focus on a comprehensive F1 FIp-FRT screen to identify both dominant and recessive mutations. Mutations that modify PTS activity will be mapped to identify the modifier gene encoded proteins. Identification of the proteins mediating PTS activities is essential for determining the molecular mechanisms of these activities and will guide future studies aimed at understanding how enhancer-promoter interactions are regulated in complex developmental genes.