Probing how transcription factors find and bind their genomic targets
Gene expression is governed by binding of transcription factor proteins to regulatory sequences in the genome, where they either recruit or block transcriptional machinery to activate or repress expression. Despite many years of research, we remain largely unable to predict where transcription factors bind in vivo, and current models cannot explain how closely related transcription factors often regulate very distinct sets of target genes. We are combining the MITOMI platform with tools from chemical biology to precisely dissect the biophysical mechanisms by which transcription factors recognize DNA and improve our ability to predict these essential interactions.
Revealing how kinetics of transcription factor binding affect occupancy and gene expression
Recent evidence suggests that non-equilibrium binding dynamics may govern transcription factor activity at regulatory sites in the genome. Despite this fact, nearly all investigations of transcription factor binding in vitro measure only binding at equilibrium, without observing kinetics. We are developing a high-throughput microfluidic assay that can measure the residency time of molecular interactions. We expect this assay will be compatible with both extracted DNA and chromatin, allowing for investigations of the native epigenetic contexts that shape gene expression.
Understanding how enzymes achieve their extraordinary catalytic efficiency and specificity
Enzymes are the most efficient catalysts known, enhancing specific reaction rates by up to 17 orders of magnitude. However, we do not fully understand how they achieve this tremendous catalytic efficiency, which limits our ability to understand their biological function and design new enzymes with industrially or medically important applications. In close collaboration with the Herschlag lab, we are using the MITOMI platform to enable high-throughput investigation of how individual residues throughout an enzyme contribute to catalysis. This new platform allows detailed biochemical characterization of thousands of enzymes in parallel, enabling structure/function studies of enzymes at unprecedented scale.
Automating printing and image acquisition and analysis
Uncovering the biophysical mechanisms that drive transcription factor binding requires the ability to efficiently and reproducibly collect and compare data from many proteins. To streamline this process, we have been automating all asepcts of the experiments, from rebuilding old microarray printers to developing new software for valve automation and image processing.