Date of Award
Doctor of Philosophy (PhD)
Proper spatiotemporal gene expression depends on specific interactions between cis-regulatory sequences (CRSs) and chromatin modifications at a genomic location. However, we do not yet understand the rules that determine how CRSs work together to influence gene expression. The goal of this thesis is to systematically assay and quantify how different genomic environments interact with CRSs to control gene expression output using a variety of genome-integrated massively parallel reporter assays (MPRAs). First, we tested the hypothesis that core promoters confer specificity by only responding to compatible enhancers and chromatin in specific genomic environments. We measured the activities of hundreds of core promoters at four genomic locations and conversely, the activities of six core promoters at thousands of genomic locations. We found that core promoters are modular components of the genome that are independently scaled up or down by diverse genomic environments. From these results, we proposed a model where genomic environments scale core promoter activities in a non-linear manner that depends on the respective strengths of the location and core promoter. Since core promoters do not appear to contribute to specific interactions in the genome, we next asked if insulators might act in a more context-specific manner. Insulators are CRSs that can block enhancers from activating target genes or act as barriers to prevent heterochromatin spreading. To test the modularity of insulators we developed MPIRE (Massively Parallel Integrated Regulatory Elements), a method that allows us to test the activities of multiple reporter genes in the same thousands of locations across the genome in parallel. Using MPIRE, we measured the activities of three insulator sequences and their mutants across thousands of genomic locations. We found that in contrast to core promoters, insulators act in a highly context-dependent manner and only function in a small number of locations across the genome. While all insulators tested demonstrated enhancer-blocking ability, only one insulator was found to have barrier activity, and insulator activity is dependent on their respective sequence motifs. The results of these two sections suggest a model where genomic environments and core promoters are broadly compatible, while insulators organize the genome to modulate specific CRS interactions. In the final section of the thesis, we asked how genomic environments control cell-to-cell variability in gene expression, or gene expression noise. To answer this question we developed SARGENT (Single-cell Analysis of Reporter Gene Expression Noise and Transcriptome), a single-cell method to quantify the variability of hundreds of reporter genes integrated across the genome in parallel. Using SARGENT, we quantified the contributions of genomic environments and cellular components to gene expression noise and identified genomic and cellular features that might regulate gene expression noise. Collectively, the results and methods in this thesis will contribute to our understanding of how genomic environments regulate various aspects of gene expression.
Chair and Committee
Barak A. Cohen
Robi D. Mitra, Ting Wang, Cristina D. Strong, Tychele N. Turner,
Hong, Clarice, "Understanding gene regulation with high-throughput genome-integrated reporter assays" (2022). Arts & Sciences Electronic Theses and Dissertations. 2798.