CAREER: Leveraging Microfluidics for High-Throughput in Vitro Investigations of Transcriptional Regulation

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All biological processes depend on the precise regulation of when genes are transcribed and how many RNA transcripts are produced. This regulation is driven primarily by transcription factor (TF) proteins that bind specific DNA sequences in the genome and then recruit additional ‘effector’ proteins to either activate or repress gene transcription. While scientists have ‘cracked’ the DNA code specifying the sequences of RNA and protein molecules produced from protein-coding DNA, it remains unclear how the TF/DNA ‘regulatory’ code that governs the strength and timing of gene expression. It is not known how closely related TFs with apparently similar DNA binding preferences recognize different sites in the genome to regulate distinct transcriptional programs. There is little known about the web of interactions between TFs and the ‘effector’ proteins required to activate transcription. This project will utilize new microfluidic technologies that enable accurate measurement of 1000s of protein/DNA and protein/protein binding interactions simultaneously and at low cost. This project will apply these technologies to better understand how TFs find and bind their DNA targets, how bound TFs recruit ‘effector’ proteins, and the degree to which the ‘regulatory code’ relies on thermodynamics. The PI will expand a hands-on microfluidics device laboratory to provide inquiry-based summer research experiences to community college students, increasing training opportunities for student populations traditionally underrepresented in STEM. Regulated gene expression is central to biology, sculpting the transformation from embryo to animal and enabling cells to respond dynamically to environmental changes. At a molecular level, this regulation is accomplished primarily by transcription factor (TF) proteins that bind DNA regulatory elements and then recruit additional protein cofactors to either activate or repress transcription. Biological diversity is simply too vast for us to ever measure TF binding and transcription in all organisms and all tissues under all conditions of interest. This project will provide in vitro measurements that quantify thermodynamic and kinetic constants of reconstituted macromolecular interactions at unprecedented scale by using multiple novel microfluidic platforms capable of measuring affinities and kinetics for up to one million protein/DNA or protein/protein interactions in parallel that were developed in the PI’s laboratory. In this project, these in vitro technologies will be used to develop quantitative and predictive models of how TFs find and bind their genomic targets and how bound TFs recruit cofactors to regulate gene expression. Using cutting-edge in silico tools, these measurements will then be integrated with existing in vivo data sets to develop quantitative models that, in turn, will be directly tested in vivo by quantifying TF and cofactor binding and gene expression for sequence variants. This project is funded by the Molecular Biophysics and Genetic Mechanisms Clusters in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.