Dissecting Gene Regulation At The Rna And Chromatin Levels

Development, homeostasis, and healthy aging depend on precise regulation of gene expression programs. As a result, a central challenge in biology is to understand, predict, and ultimately control gene expression. In the first part of the work, we leverage high-throughput biophysical data to develop quantitative models of protein-nucleic acid interactions. We apply this to Argonaute proteins, which load microRNAs (miRNAs) or small interfering RNAs (siRNAs) to form the RNA-induced silencing complex (RISC) and represses target RNA expression. We measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC, and ultimately derive quantitative models for RISC binding and target cleavage. We then show that our in vitro measurements and models predict knockdown in an engineered cellular system. We next employ a similar approach to study PUM2 binding affinity and develop a predictive model of PUM2 binding that accounts for the ensemble of possible PUM2 binding modes. We then measure equilibrium binding of PUM2 to structured RNAs and demonstrate that RNA secondary structure can facilitate the formation of cooperative interactions between RNA-binding proteins. Finally, we expand on this work to develop a quantitative description of the DNA guided bacterial proteins TtAgo and Cas9, which have emerged as tools for the control of gene expression. In the second part of this work, we shift to studying gene regulation in normal human colon and during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC). We collect single-cell chromatin accessibility and transcriptomic measurements and find that a large fraction of polyp and CRC cells exhibit a stem-like phenotype. We define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from normal to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T-cells, and a subtype of pre-cancer associated fibroblasts. In the cancerous state, we observe T-cell exhaustion, RUNX1-regulated cancer associated fibroblasts, and increasing accessibility associated with HNF4A motifs in epithelia. Finally, we show that methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.