Systems and synthetic biology approaches enable first-principles models of transcription in human cells
Abstract/Contents
- Abstract
- The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF's capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. Chapters 2-4 provide a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration. Chapter 5 demonstrates how novel large serine recombinases can facilitate highly controlled and fast parallel DNA reporter assays by integrating linear DNA library members into landing pads. Chapter 6 characterizes how transcriptional run-on between two tandem genes can lead to unexpected gene expression dynamics in synthetic gene systems. Taken together, this thesis provides novel biological insights and tools to advance understanding and engineering of gene expression in human cells.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2024; ©2024 |
| Publication date | 2024; 2024 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Hinks, Michaela Marie |
|---|---|
| Degree supervisor | Bintu, Lacramioara |
| Degree supervisor | Greenleaf, William James |
| Thesis advisor | Bintu, Lacramioara |
| Thesis advisor | Greenleaf, William James |
| Thesis advisor | Fordyce, Polly |
| Degree committee member | Fordyce, Polly |
| Associated with | Stanford University, School of Engineering |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Michaela Marie Hinks. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2024. |
| Location | https://purl.stanford.edu/tb570zc6683 |
Access conditions
- Copyright
- © 2024 by Michaela Marie Hinks
- License
- This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
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