Regulation of Gene Expression Through Nucleic Acid Binding Proteins: New Paradigms, Perspectives, and Tools
Wolfe, Michael
2019
Abstract
More than 50 years ago, Jacob and Monod first laid out a model for the regulation of gene expression through the interaction of proteins and nucleic acids. With the advent of high-throughput sequencing and the unprecedented ability to take millions of simultaneous measurements of the same biological system, better understanding of the full complexity of gene regulation is finally being unraveled. Here, I present my graduate work investigating the control of gene expression at the transcriptional and post-transcriptional levels through the analysis of high-throughput biological measurements in bacterial and human cell culture. In the realm of bacterial transcriptional control, I explore the impact of a global regulator, Lrp, and its regulation of up to one third of the genes in E. coli. We identify a prevalent mode of "poised" binding, where Lrp is bound at a given promoter but only appears to have a regulatory effect under certain conditions. We find that Lrp appears to change its binding mode from a non-specific A/T-rich preference in early growth phases to a more sequence specific preference in later growth phases. I also look at promoter-independent effects on transcriptional regulation in E. coli. I describe key features of the bacterial genome that predict the position-dependent effect of transcription on a randomly integrated uniform reporter gene. We find that binding signal from the highly abundant nucleoid associated proteins H-NS and Fis act as key predictors for low and high transcription, respectively. We also find that integration proximity to a ribosomal RNA operon appears to be the single greatest contributor to position-dependent transcriptional activation. Absent of ribosomal RNA operon effects, we find that recent maps of E. coli chromosomal structure do not help us explain the periodic transcriptional signal obtained from our library. In eukaryotic systems, I focus on post-transcriptional control through the regulation of mRNA decay. I review methods that can be used to measure the decay of mRNA in a high-throughput manner. I highlight the importance of spike-in controls and demonstrate strategies to determine relative mRNA decay between experimental conditions at minimal cost. Finally, I present my work identifying key sequence features that allow for the prediction of mRNA decay mediated by the human Pumilio proteins. I find that contextual sequence features around predicted PUM binding sites contribute meaningful information to the prediction of PUM-mediated post-transcriptional regulation. I also demonstrate that human Pumilio proteins primarily modulate RNA abundance through controlling mRNA decay and not through control of transcription. Taken together, my graduate work provides a comprehensive view of the regulation of gene expression at both transcriptional and post-transcriptional levels.Subjects
gene regulation transcription RNA decay RNA binding protein transcription factor global regulator
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