Dissecting the functions of a novel cis element PTS

描述（由申请人提供）：我们已经从drosophht Bithorax复合物中确定了一个新的顺式调控元件，启动子靶向序列（PTS）。PTS具有抗绝缘子活性，允许被阻断的增强子通过中间绝缘子激活其启动子；它也有一个启动子靶向活动，选择性地只针对两个可能的启动子中的一个。PTS位于腹部- b （Abd-B）基因中，其中调控区域由诸如Fab-7和Fab-8之类的绝缘子dna组织成片段特异性结构域。PTS的突变导致Abd-B表达的丧失和腹部的同质转化。我们提出PTS通过克服Fab绝缘子的增强子阻断活性来介导增强子-启动子相互作用，从而将Fab绝缘子转化为局部域边界元。我们的工作假设是，PTS的功能是通过一种机制在增强子周围的DNA和启动子附近的DNA之间形成稳定的联系，这种机制对绝缘子阻断不敏感，表观遗传稳定，并且独立于与增强子相互作用的激活子。考虑到果蝇和脊椎动物之间Hox基因簇的惊人相似性，我们有理由相信PTS代表了一类新的顺式调控元件，可以调节无脊椎动物和脊椎动物Hox基因簇中的远程增强子-启动子相互作用。我们建议通过检验与我们的“稳定关联模型”相关的几个假设来剖析PTS的功能。在特异性Aim la中，我们将测试一个可遗传的稳定增强子-启动子相互作用的形成与增强子身份和增强子结合蛋白无关的预测。在lb中，我们将测试PTS是否使绝缘子失活，以及绝缘子是否有助于PTS的启动子靶向活性。最后，在Aim lc中，我们将确定PTS与增强子、启动子和绝缘子的相对位置如何影响其抗绝缘子和靶向启动子的活性，以及PTS如何影响Abd-B中增强子-启动子特异性。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元件起作用的基因或蛋白质。我们将主要关注一个全面的F1 FIp-FRT筛选，以确定显性和隐性突变。修饰PTS活性的突变将被定位，以识别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至关重要，并将指导未来的研究，旨在了解增强子-启动子相互作用如何在复杂的发育基因中被调节。

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.