Date of Award
Doctor of Philosophy (PhD)
Genomics & Computational Biology
Arjun . Raj
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.
Mellis, Ian Alexander, "Systems Biology Of Gene Regulation Across Scales: From Single Molecules To Cellular Identities" (2019). Publicly Accessible Penn Dissertations. 3594.