Systems Biology Of Gene Regulation Across Scales: From Single Molecules To Cellular Identities

Gene regulation takes many forms and is responsible for phenotypes at the scale of individual molecules up through the scale of complex tissue functions. At the smallest level, single-base modifications of individual mRNA molecules transcribed from the same gene can lead to functionally different protein products. In the first chapter of this thesis, I develop a new method, inoFISH, and associated analytical tools to visualize and quantify RNA editing with single molecule resolution in single mammalian cells. Using this new method in conjunction with mathematical modeling I show that the heterogeneity of single-cell mRNA editing rates across a population depends on the gene of interest. Further, I characterize subcellular localization patterns of edited and unedited mRNAs. At the other end of the spectrum, the regulation of transcriptome-wide patterns of gene expression can underpin cellular identities. In the second chapter of this thesis I develop a new experimental design and analytical framework for prioritizing lists of transcription factors that can be used for directed changes of cellular identity. With Perturbation Panel Profiling (P3), I show that cardiomyocyte lineage-driving transcription factors are more frequently up-regulated, or “perturbable”, than other highly expressed transcription factor genes. I subsequently demonstrate that a known cocktail of cardiomyocyte-perturbable transcription factors enables cardiac transdifferentiation of several types of human fibroblasts. Lastly I extend perturbability-based selection of transcription factors to another biological context, i.e., fibroblast reprogramming to pluripotency. I show that fibroblast-perturbable factor knockdown often enables more efficient fibroblast reprogramming. Together, my thesis makes critical steps toward understanding and engineering gene regulation through the development of a diverse array of methods, experimental designs, and analytical frameworks.