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

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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.