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TRANSCRIPTION FACTOR-DNA INTERACTIONS: INVESTIGATING BINDING SPECIFICITIES IN BASE-RESOLUTION MODELS

Author
Lee, Seungha Alisa
Abstract
The epigenome is a multitude of all molecular interactions that governs genome regulatory activities. Sequence-specific transcription factors (TFs) are the key regulators that control gene expression by binding to specific DNA sequences at promoters or distal enhancers. Understanding how TFs recognize their DNA binding sites forms the basis for understanding mechanisms of transcriptional control. Technological developments have led to the identification of TF binding preferences and revealed how TF-DNA interactions, local DNA structure, and genomic features influence TF-DNA binding. Yet, a precise human epigenome remains largely undefined. Mechanistic insights into how transcription factors recognize their cognate sites across a genome may be best understood through a combination of in vivo and in vitro genome-wide binding measurements. To this end, I provided the first single base pair measurements of human stem cell factor KLF4 binding to a noncognate DNA sites in vivo. To further explore the mechanistic basis for such interactions, I initiated development of an in vitro biochemically defined system for measuring protein-DNA interactions genome-wide for human TFs. In the first stage of this process, I developed methods to express human TFs using in vitro transcription/ translation systems as well as an E. coli T7 expression system. By using His-GFP fusions to these TFs, I tracked their production and purification in real time using GFP’s intrinsic fluorescence. I then explored whether His-GFP can be used to establish an in vitro genome-wide assay for site-specific DNA binding (PB-exo assay), where nickel resin is used to retain His-tagged proteins, and GFP is used track binding in real time. Collectively, I demonstrated the feasibility of in vitro protein expression and purification as applied towards the PB-exo assay, including using nickel resin in place of antibodies to immunoprecipitated target proteins. The work here provides several concrete steps towards developing a genome-wide assay for in vitro protein-DNA interactions of human TFs.
Description
50 pages
Date Issued
2021-12Subject
ChIP-exo; DNA-binding specificity; PB-exo; TF-DNA interactions
Committee Chair
Pugh, Frank
Committee Member
Grimson, Andrew William; Kwak, Hojoong
Degree Discipline
Genetics, Genomics and Development
Degree Name
M.S., Genetics, Genomics and Development
Degree Level
Master of Science
Type
dissertation or thesis