Dissecting the functions of a novel cis element PTS

剖析新型顺式元件 PTS 的功能

• 批准号: 6574101
• 负责人: JUMIN ZHOU
• 金额: $ 29.52万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6574101
• 关键词: Drosophilidae; arthropod genetics; chromatin; enhancer binding protein; fluorescence microscopy; genetic enhancer element; genetic models; genetic promoter element; genetic regulatory element; genetic screening; globin; homeobox genes; immunoprecipitation; intermolecular interaction; molecular cloning; reporter genes

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.

描述（由申请人提供）：我们已经从 Drosophiht Bithorax 复合物中鉴定出一种新的顺式调控元件，即启动子靶向序列（PTS）。 PTS具有抗绝缘体活性，允许原本被阻断的增强子在干预绝缘体上激活其启动子；它还具有启动子靶向活性，选择性地仅靶向可能的启动子中的一个。 PTS 位于 Abdominal-B(Abd-B) 基因中，其中调节区域通过 Fab-7 和 Fab-8 等绝缘子 DNA 组织成节段特异性结构域。 PTS 突变导致 Abd-B 表达缺失和腹部同源异型转化。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素。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 之间形成稳定的关联来发挥作用，这种机制对绝缘体块不敏感，表观遗传稳定，并且独立于与增强子相互作用的激活剂。鉴于果蝇和脊椎动物之间的Hox簇具有惊人的相似性，我们有理由相信PTS代表了一类新的顺式调节元件，其调节无脊椎动物和脊椎动物的Hox基因簇中的远程增强子-启动子相互作用。我们建议通过测试与我们的“稳定关联模型”相关的几个假设来剖析 PTS 的功能。在特定目标1a中，我们将测试可遗传的稳定增强子启动子相互作用的形成独立于增强子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启动子靶向活性，以及​​ PTS 如何影响 Abd-B 中的增强子-启动子特异性。在第二个具体目标中，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，我们将进行遗传筛选，以分离通过 PTS 元件发挥作用的基因或蛋白质。我们将主要关注全面的 F1 FIp-FRT 筛查，以识别显性和隐性突变。改变 PTS 活性的突变将被映射以识别修饰基因编码的蛋白质。介导 PTS 活性的蛋白质的鉴定对于确定这些活性的分子机制至关重要，并将指导未来旨在了解复杂发育基因中增强子-启动子相互作用如何调节的研究。