High-throughput and single-cell methods for measuring gene expression and chromatin modification changes produced by viral proteins in human cells
Abstract/Contents
- Abstract
- Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression and chromatin state. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription, which specific sequences contribute to this function, and how these sequences alter the underlying chromatin. In this dissertation, I demonstrate how high-throughput synthetic biology and genomics can address these questions via functional and mechanistic investigation at scale. First, using a high-throughput assay, I measure the transcriptional regulatory potential of over 60,000 protein tiles across more than 1,500 proteins from 11 coronaviruses, all nine human herpesviruses, and other viruses that infect humans. These efforts massively expand viral protein annotations through the discovery of hundreds of new transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, effector domains in completely uncharacterized proteins from highly ubiquitous viruses, dual activation-repression domains in viral interferon regulatory factors, and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in a system with live virus. For the effector domains identified, I employ high-throughput mutagenesis to pinpoint sequence motifs essential for function and chemical inhibition screens to identify domains that are dependent on the activities of major chromatin-modifying enzymes. In addition to discovering viral effector domains and their potential interaction partners, we need to understand how they change the chromatin state of their target genes. As such, I next review myriad methods to map chromatin modifications, with a focus on single-cell approaches that can measure heterogeneity such as the kind we observe across many viral transcriptional effectors. I discuss recent advances that enable single-cell measurements, including optimization to reduce DNA loss, improved DNA sequencing, barcoding, and antibody engineering. Subsequently, I apply two existing methods to measure chromatin modification changes produced by well-characterized human chromatin regulators upon transcriptional silencing and demonstrate the ability of these methods to quantify the spread of classically associated histone and DNA modifications. Finally, I propose novel methods for the simultaneous measurement of multiple chromatin modifications at the single-cell level to investigate how these modifications change in relation to one another. I discuss the promise these methods hold for characterizing viral protein-induced changes to chromatin, which will be critical for studying their biological implications and for developing new compact gene regulation tools.
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 | 2023; ©2023 |
| Publication date | 2023; 2023 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Ludwig, Connor Hayakawa |
|---|---|
| Degree supervisor | Bintu, Lacramioara |
| Thesis advisor | Bintu, Lacramioara |
| Thesis advisor | Covert, Markus |
| Thesis advisor | Qi, Lei, (Professor of Bioengineering) |
| Degree committee member | Covert, Markus |
| Degree committee member | Qi, Lei, (Professor of Bioengineering) |
| Associated with | Stanford University, School of Engineering |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Connor Hayakawa Ludwig. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2023. |
| Location | https://purl.stanford.edu/br206pg3647 |
Access conditions
- Copyright
- © 2023 by Connor Hayakawa Ludwig
- License
- This work is licensed under a Creative Commons Attribution Non Commercial No Derivatives 3.0 Unported license (CC BY-NC-ND).
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