Uncovering the fundamental principles of transcriptional regulation

/Abstract Cells are required to turn specific genes on, off, up or down in response to stimuli. Transcription factors (TFs) are largely responsible for tuning (regulating) gene expression by binding to specific sites on DNA to interact with (inhibit or enhance) the transcriptional machinery. While it is now possible to precisely measure the abundance of these molecules, where they interact and the resulting level of expression from a target gene, we are still unable to predict resulting levels of gene expression from the regulatory sequence of a given gene. This is largely due to the prevalence of complicating factors that simultaneously impact regulation; each natural gene tends to be regulated by one or more TF species acting on it simultaneously and each TF has tens to thousands of binding sites in the genome. The aim of this project is to systematically measure the function and occupancy of each TF in the model bacterium E. coli in order to parameterize a predictive model of gene regulation. This novel approach exploits a tight interplay between predictive theory and quantitative experimental measurements. We will achieve this goal by using a library of E. coli strains, created previously in my lab, where the concentration of any TF can be precisely induced and measured. This data will be interpreted through a biophysical model of gene expression to characterize the regulatory function of every transcription factor as it binds to the gene and its occupancy at binding sites throughout the genome. Importantly, based on this characterization process, our model predicts regulation of more complex scenarios such as promoters regulated by multiple TFs or expressed from different RNA Polymerase assemblies. Through this process we will reveal the basic features of gene regulation by single TFs in E. coli and test how this fundamental knowledge can be assembled into a more complete model of gene regulation. In the next 5 years, we will use these measurements the function and concentration-occupancy relationship for each TF in E. coli in order to build and refine our model. We will then use that model to test its predictions against organism-wide gene regulation patterns measured at different TF concentrations. Project Number: 1R35GM161523-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Robert Brewster | Institution: UNIV OF MASSACHUSETTS MED SCH WORCESTER, WORCESTER, MA | Award Amount: $460,625 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-J (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260548