A novel method to characterize cis-regulatory complexes during development

项目摘要/摘要 基因精确的时间和空间转录是大多数发育的基本组成部分 多细胞生物体中的过程。尽管很重要，但关键的细节，如蛋白质的身份 在其天然细胞环境中占据特定顺式调控元件的基因通常是未知的。此外，还有 需要一种方法来有力地评估顺式调控元件中的特定序列要求 这是DNA-蛋白质相互作用所必需的。因此，在确定 基因调控网络的蛋白质和DNA组成。这个项目的长期目标是开发一种 一种稳健、无偏倚的方法，用于鉴定在其天然中占据特定顺式调控元件的蛋白质 蜂窝环境。这一提议的中心假设是用于DNA导入的电穿孔方法 将允许对相关转录因子的高灵敏度检测，也是一种实验途径 评估顺式调控元件的序列要求。进行这项研究的理由是 在发育过程中缺乏关于受调控转录的基本信息 和疾病。这里描述的方法将建立一个平台，可以用来克服这一关键问题 这是一种障碍，可以适应不同的范式。为了实现这一目标，以下两个目标 建议：1)构建共价结合生物素转运体的修饰顺式调控元件载体 对DNA和2)用修饰的顺式调节元件质粒标记蛋白质的评价。至 为评估这项技术提供了基础，这些目标将使用先前建立的电穿孔 脊椎动物视网膜发育中的范例和已知结合位点的顺式调节元件 转录因子。第一个目标将使用标准的DNA和蛋白质修饰程序来生成顺式- 调控元件报告质粒具有生物素化蛋白质的能力，靠近顺式- 监管要素。第二个目标将决定这一方法的有效性，以标记已知的 转录因子相互作用因子使用技术来直接评估这些特定的蛋白质以及 无偏质谱方法，以确定这种无偏技术的灵敏度，以识别这些 已知的DNA-蛋白质相互作用。这种方法是创新的，因为它将使用顺式监管元素目标 使用共价结合的生物素化试剂，这些试剂可能提高检测蛋白质的灵敏度 互动者。此外，与目前的技术形成对比的是，拟议的系统将允许 一种简便的检测转录因子招募中特定DNA序列要求的方法 设计严格的控制措施以确认互动。在拟议的项目完成时，一部小说 可用于在天然细胞环境中识别DNA-蛋白质相互作用的方法将是 开发和验证。这种方法的应用有望提供对关键参数的深入了解 顺式调控对我们理解发育机制和疾病病因学的影响。

PROJECT SUMMARY/ABSTRACT The precise temporal and spatial transcription of genes is a fundamental component of most developmental processes in multicellular organisms. Despite its importance, critical details such as the identity of the proteins that occupy specific cis-regulatory elements in their native cellular context are often unknown. In addition, there is a need for methods to robustly evaluate the specific sequence requirements within cis-regulatory elements that are required for DNA-protein interactions. Thus, there exist critical barriers to the identification of the protein and DNA components of gene regulatory networks. The long-term goal of this project is to develop a robust, unbiased method for identification of proteins that occupy specific cis-regulatory elements in their native cellular context. The central hypothesis of this proposal is that the electroporation method for DNA introduction will allow for high sensitivity detection of associated transcription factors and also an experimental avenue to evaluate the sequence requirements of cis-regulatory elements. The rationale to undertake this study is that there is a lack of fundamental information with regards to regulated transcription in the context of development and disease. The approach described here will establish a platform that can serve to overcome this critical barrier and can be adapted to diverse paradigms. To accomplish this goal, the following two aims are proposed: 1) Creation of modified cis-regulatory element plasmids with biotin transfer agents covalently bound to DNA and 2) Evaluation of biotin-labeling of proteins with modified cis-regulatory element plasmids. To provide a basis to evaluate this technique, these aims will use a previously established electroporation paradigm in the developing vertebrate retina and a cis-regulatory element with defined binding sites for known transcription factors. The first aim will use standard DNA and protein modification procedures to generate cis- regulatory element reporter plasmids with the ability to biotinylate proteins in close proximity to the cis- regulatory element. The second aim will determine the effectiveness of this approach to label the known transcription factor interactors using techniques to assess directly these specific proteins as well as an unbiased mass spectrometry approach to determine the sensitivity of this unbiased technique to identify these known DNA-protein interactions. This approach is innovative because it will use cis-regulatory element targets with covalently associated biotinylation agents which are likely to increase the sensitivity to detect protein interactors. In addition, in contrast to current technologies, the proposed system will allow for a robust and facile method to examine the requirements of specific DNA sequences in transcription factor recruitment and to design rigorous controls to confirm interactions. At the completion of the proposed project, a novel methodology that can be used to identify DNA-protein interactions in a native cellular context will have been developed and validated. The application of this method is expected to provide insights into critical parameters of cis-regulation with implications for our understanding of developmental mechanisms and disease etiology.