EAGER: Development of a yeast synthetic reporter system to measure activity of transcription factor complexes

在生物学中，通常的情况是相对简单的部分可以互换地组合在一起，从而获得令人惊讶的复杂结果。一个众所周知的例子是DNA编码，它只有四个独特的核苷酸，但可以组合在一起，创造出我们在地球上看到的所有生命的复杂性。另一个例子是潜在的无休止的蛋白质组合--通常组装成分子机器的小分子--它们被用来调节生物过程。许多科学家特别感兴趣的是被称为转录因子的特殊蛋白质组是如何聚集在一起调控基因表达的。这一点很重要，因为这些转录因子控制的基因表达的变化是对环境变化的大多数反应的基础。在试管和活体中，已经开发了许多技术来研究转录因子如何调节基因表达，但仍然缺乏实验系统，允许科学家检查一组相互作用的转录因子如何在活细胞中组装和发挥作用。因此，该项目将开发一种尖端系统，使科学家能够同时表达三种(或更多)蛋白质，并解决这个最初的问题--这些相对简单的转录因子如何共同作用，导致基因表达的复杂变化？除了为科学界提供有价值的工具外，该项目还将对本科生产生教育和培训影响，他们将通过强调规划、执行和数据分析的教学模块接触研究。研究生和博士后研究员将进行这项研究，这将为他们提供开发一种新的生物学研究系统的培训机会和智力挑战。该项目还将在每年夏天让一名当地高中生作为研究团队的一部分。多亚基转录因子如何实现基因组水平的底物亲和力并调控基因表达在很大程度上尚不清楚。研究转录因子功能的系统通常依赖于体外分析，这种方法具有分辨率高的优点，但高度专业化，往往需要蛋白质纯化。或者，各种体内分析可以提供局部或全局结合模式，但通常分辨率较低。在这里，一个新的体内酵母系统-它包括高度特异的激素诱导，一个新的效应盒，和一个荧光素酶报告系统-将被开发来检测多亚基转录因子和它们可能的顺式调控元件之间的相互作用。一旦开发出来，该系统将允许快速和准确地评估任何转录因子亚单位或其同源顺式调控元件的变化如何改变结合亲和力和基因表达输出。对于概念验证研究，将检查植物(拟南芥)核因子Y(NF-Y)与Constans、Constans-like和CAB定时(CCT)转录因子之间的相互作用。一旦得到证实，这种酵母系统的模块化性质将使其很容易被其他实验室使用。

In biology, it is often the case that relatively simple parts can be interchangeably combined to achieve surprisingly complex outcomes. A well-known example is the DNA code, which has only four unique nucleotides, but can be combined to create all of the living complexity we see on planet earth. Another example is the potentially endless combinations of proteins - small molecules that often assemble into molecular machines - that are used to regulate biological processes. Of particular interest to many scientists is how special groups of proteins called transcription factors come together to regulate gene expression. This is important because changes in gene expression, as controlled by these transcription factors, underlie most responses to environmental change. Numerous techniques, both in the test-tube and in living organisms, have been developed to examine how transcription factors regulate gene expression, but there remains a paucity of experimental systems that allow scientists to examine how groups of interacting transcription factors assemble and function in living cells. Therefore, this project will develop a cutting-edge system to allow scientists to simultaneously express three (or more) proteins and address this initial question - how do these relatively simple transcription factors work together to result in complex changes of gene expression? In addition to providing a valuable tool for the scientific community, the project will have education and training impact for undergraduates who will be exposed to research via a teaching module emphasizing planning, execution, and data analysis. Graduate students and postdoctoral fellows will carry out the research, which will provide them with the training opportunity and intellectual challenge of developing a novel system for biological research. The project will also involve a local high school student as part of the research team in each summer.How multi-subunit transcription factors achieve genomic-level substrate affinity and regulate gene expression is largely unknown. Systems for studying transcription factor function often rely on in vitro assays that can have the advantage of excellent resolution, but are highly specialized and tend to require protein purification. Alternatively, a variety of in vivo assays can provide either local or global binding patterns, but typically suffer from low resolution. Here a novel in vivo yeast system - which incorporates highly-specific hormone induction, a novel effector cassette, and a luciferase reporter system - will be developed to examine the interactions between multi-subunit transcription factors and their putative cis regulatory elements. Once developed, the system will permit rapid and accurate evaluation of how changes in any of the transcription factor subunits, or their cognate cis regulatory elements, alter binding affinity and gene expression outputs. For proof-of-concept studies, interactions between plant (Arabidopsis thaliana) NUCLEAR FACTOR Y (NF-Y) and CONSTANS, CONSTANS-LIKE, and TIMING OF CAB (CCT) transcription factors will be examined. Once proven, the modular nature of this yeast system will make it readily adaptable for use by other labs.