Only a fraction of the over 20,000 genes present in every cell of the body are used at a given time. The choice of which genes to use is determined by environmental cues, such as heat, cold, food, hormones, and injury. This project studies how proteins function to turn genes on and off in response to hormones. Understanding this problem requires expertise from a variety of different disciplines, including physics, chemistry, and biology, that are often separate in the scientific culture. This research fosters cross-disciplinary training early, at the undergraduate level, by re-engineering how organic chemistry laboratory is taught, and connecting it to this pressing research problem. The results of this study provide critical insight into how cells respond to their environment while preparing the next generation of scientists to break down interdisciplinary barriers in the workplace.
Proteins called transcription factors (TFs) have the critical task of integrating intra- and extracellular signaling pathways to precisely regulate gene expression in response to stimuli. TFs regulate genes by binding specific DNA sequences and nucleating the machinery needed for transcription (RNA synthesis). Recent large-scale efforts have defined the core DNA sequence preferences for hundreds of TFs, but very little is known about how these crucial regulatory signals change TF:DNA binding activity. This project may determine how external signals remodel the intrinsic properties of TF:DNA binding to redirect genomic association and gene regulation. Using the glucocorticoid receptor (GR, a hormone activated TF) as a model system, the effect of ligands and phosphorylation on the specificity and kinetics of DNA binding are measured in vitro, and compared to GR genomic occupancy and DNA binding dynamics in live cells. DNA binding specificity are measured in vitro using high resolution systematic evolution of ligands by exponential enrichment (SELEX-seq) from which thermodynamic models of binding covering all possible sequences are generated, and in cells using chromatin-immunoprecipitation followed by deep sequencing (ChIP-seq). DNA binding kinetics are measured in vitro using single molecule total internal reflection fluorescence microscopy (TIRFM) and in cells using single molecule single molecule tracking (SMT). The consequence of changing specificity and kinetics are then tested using cell lines with engineered binding sites generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats). Students in the undergraduate Advanced Organic Laboratory course at Butler University synthesize some of the ligands to be used in the research, and also participate in their testing. The results build on current static descriptions for understanding specificity and regulation, by enabling creation of new models that predict the effect of changing intrinsic properties on regulating cellular function.
This project is co-funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences and the Division of Emerging Frontiers in the Biological Sciences Directorate, and by the Chemistry of Life Processes Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.