PROJECT SUMMARY Despite having limited sets of signaling components and number of genes, cells must be able to distinctly respond to a large number of input signals, such as environmental stresses, as well as execute diverse gene expression programs. It is vital that cells receive, transmit, filter, and act upon these signals accurately to trigger the appropriate downstream gene response program, as dysregulation and aberrant signaling have important implications in the initiation or progression of diseases such as immune disorders and cancer. Specifically, a single transcription factor (TF) can respond to a wide variety of input signals by changing its nuclear localization dynamics to activate specific promoters. However, the study of transcriptional dynamics is limited to largely correlational relationships due to technical barriers such as cell-to-cell variability, pleotropic effects of experimental perturbations, expression averages that mask heterogeneity, and fluorescence based techniques that limit the number of measurable target genes. The central hypothesis of this work is that the functional connections between TF dynamics and their downstream regulation of gene expression programs can be identified and tuned by the integrated use of carefully characterized and controlled optogenetic systems with single cell RNA sequencing (scRNAseq), increasing our understanding of gene regulation in human cells for engineering and biomedical applications. NF-κB (p65/RELA), which regulates hundreds of genes and is heavily implicated in immunological responses and cancer, will serve as the model TF for our system. To address this challenge and information gap, the proposed work will build upon a suite of sophisticated tools developed in our lab to combine optogenetic based TF translocation with scRNAseq. We will establish a robust, tightly controlled system and examine the genome wide effect of direct perturbations of NF-κB across thousands of cells. Three main objectives will be targeted: Aim 1 will result in the development of a fully controllable, optogenetic system to precisely regulate p65 translocation in HeLa cells by landing pad integration, optimization of localization dynamics, and validation by reverse transcription quantitative polymerase chain reaction of known target genes. Aim 2 will focus on determination of whole-genome gene transcriptomic profiles and cell-to-cell heterogeneity in response to traditionally used, chemically stimulated p65 dynamics using scRNAseq. By combining these two components, Aim 3 will directly control p65 dynamics with 15 distinct optogenetic inputs and assess resultant single cell transcriptomic changes using scRNAseq. In sum, we aim to provide an efficient, functional system to provide direct, causal relationships between TF dynamics and gene expression in human cells and exact control in cellular engineering by leveraging complete control of TF translocation through optogenetics, precise tools in mammalian s...