Uncovering the fundamental principles of transcriptional regulation

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Project summary/Abstract The regulatory code, inscribed in DNA, specifies how the expression of a gene will be tuned by transcription factors (TFs) in response to an environmental or intracellular stimulus. This code sets how each individual gene responds to stimuli by controlling when and where TFs bind. In my lab, our work is dedicated to developing a predictive understanding of gene expression. This is accomplished through a close interplay between quanti- tative theoretical predictions based on detailed biophysical models, and an experimental approach guided by falsifiable predictions for the quantitative consequence of systematic perturbation applied to a synthetic target gene. The work in my lab, which uses E. coli as a model organism, focuses on the systematic dissection of two distinct roles of regulatory binding sites in gene expression: The regulatory role of individual, local “cis-regulatory” binding sites. The regulatory role of non-local, competing binding sites scattered throughout the genome. To characterize local, cis-regulatory interactions we use a synthetic biology approach to systematically measure the regulatory function of every TF as a function of where on the gene it binds and to what sequence it binds. Through this process we will uncover the isolated function of each TF in the absence of the gene-specific factors (such as feedback and TF-TF interactions) that occlude this basic information in genomic data. To study the role of competing binding sites, we use this same synthetic biology approach to control the number and strength of TF binding sites to measure how competition for TFs controls spatial and temporal patterns in gene expression. Taken together, these directions of investigation are aimed at providing a complete picture of regulation at the level of a single gene. These methods are not orthogonal, it is difficult to study one without appreciating the other; to quantify how competition alters expression we must understand the “isolated” regulation of the local regulatory elements acting alone, and to study local regulatory elements we must understand the impact of the unavoidable, naturally occurring competing binding sites around the genome. Our approach is designed to isolate and quantify these regulatory effects to provide the foundation required to predict expression from the complex regulatory schemes observed in natural genes. In the next 5 years, we will demonstrate that by characterizing individual TF function based on binding location and sequence, we can disentangle the role of gene-specific features from basic TF function in order to under- stand how these components act together in the complex regulatory regions seen in naturally occurring genes. Furthermore, we will develop a theoretical model that accounts for the role of TF competition in orchestrating expression patterns in space and time that are crucial for cellular decision making and fitness.