Investigating the Spreading of Gene Silencing and Reactivation through the Chicken HS4 Insulator
Abstract/Contents
- Abstract
- Chromatin modifications help control gene expression in mammalian cells and spread through positive feedback mechanisms. This spreading can help coordinate gene expression programs, but can also lead to accidental silencing of transgenes. Insulators are genetic elements that can protect transgenes from background silencing. It is hypothesized that they act as barriers against heterochromatin spreading. However, the spreading of heterochromatin-induced gene silencing across insulators has not been directly tested. By engineering Chinese Hamster Ovary (CHO) cells with site-specifically integrated dual reporters, we developed a method to measure the spreading of gene silencing and reactivation across insulators. Each of the dual reporters contains two genes expressing fluorescent proteins separated by different insulator geometries containing the most widely used eukaryotic insulator, HS4, and its core region. Using a doxycycline-inducible system, chromatin regulators associated with histone methylation (KRAB, EED) and DNA methylation (DNMT3B) were recruited for five days at varying strengths to the upstream gene to induce gene silencing. The cells were monitored for their silencing and reactivation dynamics using flow cytometry. Surprisingly, neither the HS4 insulator nor its core region prevented spreading of silencing from the upstream target gene to the downstream gene. However, the presence of insulators hindered the ability to silence both genes when compared to spacer DNA. Moreover, after the end of EED and KRAB recruitment, the reactivation rates of both genes were 2-3x higher when the upstream gene was flanked by core insulators. This element also led to reactivation of both genes after DNMT3B silencing, despite previous reports that DNA methylation leads to permanent epigenetic memory. Therefore, rather than acting as a barrier to spreading, the HS4 insulator activates the entire gene locus.
Description
| Type of resource | text |
|---|---|
| Date created | May 2019 |
Creators/Contributors
| Author | Herschl, Michael |
|---|---|
| Primary advisor | Bintu, Lacramioara |
| Degree granting institution | Stanford University, Department of Bioengineering |
| Advisor | Covert, Markus |
Subjects
| Subject | chromatin |
|---|---|
| Subject | insulators |
| Subject | spreading |
| Subject | epigenetics |
| Subject | HS4 |
| Subject | Department of Bioengineering |
| Genre | Thesis |
Bibliographic information
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- Use and reproduction
- User agrees that, where applicable, content will not be used to identify or to otherwise infringe the privacy or confidentiality rights of individuals. Content distributed via the Stanford Digital Repository may be subject to additional license and use restrictions applied by the depositor.
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Preferred citation
- Preferred Citation
- Herschl, Michael. (2019). Investigating the Spreading of Gene Silencing and Reactivation through the Chicken HS4 Insulator. Stanford Digital Repository. Available at: https://purl.stanford.edu/jy909ps5209
Collection
Undergraduate Theses, School of Engineering
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- Contact
- mherschl@alumni.stanford.edu
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