Systematic perturbation of retroviral long terminal repeats reveals widespread long-range effects on human gene regulation
Abstract/Contents
- Abstract
- Recent work suggests the extensive adaptation of transposable elements (TEs) for host gene regulation. However, the high numbers of integrations typical of TEs, coupled with sequence divergence within families, have made the systematic interrogation of the regulatory contributions of TEs quite challenging. I employed chimeric arrays of gRNA oligos (CARGO), our recently developed method for CRISPR guide RNA (gRNA) multiplexing, to facilitate the targeting of LTR5HS, an ape-specific class of HERVK (HML-2) long terminal repeats (LTRs) that is transcriptionally active during early human development and present in ~700 copies throughout the human genome. I combined CARGO with CRISPR activation (CRISPRa) to induce or CRISPR interference (CRISPRi) to silence LTR5HS en masse in human embryonal carcinoma cells, enabling strong control of RNA levels of LTR5HS, and both RNA and protein levels of HERVK proviral genes. This system robustly and specifically targeted the vast majority of LTR5HS insertions, while critically excluding non-HERVK (HML-2) TE sequences. CRISPRa and CRISPRi also affected the chromatin state at LTR5HS insertions by manipulating histone tail modifications, with CRISPRa causing the deposition of active chromatin marks, and CRISPRi triggering the removal of active chromatin marks and the addition of repressive marks instead. Remarkably, activation and silencing of LTR5HS were associated with massive changes in the transcriptional profile of these cells, with thousands of genes affected. Most importantly, CRISPRa and CRISPRi reciprocally up- and down-regulated a set of 275 genes, termed LTR5HS-regulated genes, including many genes that are expressed in the developing human blastocyst concurrently with the LTR5HS insertions themselves. Interestingly, genes controlled by LTR5HS are more highly expressed in the developing blastocyst of humans compared to rhesus, an old-world monkey that predates the infection by HERVK (HML-2) LTR5HS. Expression of LTR5HS-regulated genes in human cells was strongly dependent on the presence of specific LTR5HS retroviral sequences located up to hundreds of kilobases away, consistent with a pervasive function of LTR5HS elements as early embryonic enhancers in apes.
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 | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Fuentes, Daniel Roberto |
|---|---|
| Degree supervisor | Wysocka, Joanna, Ph. D |
| Thesis advisor | Wysocka, Joanna, Ph. D |
| Thesis advisor | Fuller, Margaret T, 1951- |
| Thesis advisor | Jarosz, Daniel |
| Thesis advisor | Wernig, Marius |
| Degree committee member | Fuller, Margaret T, 1951- |
| Degree committee member | Jarosz, Daniel |
| Degree committee member | Wernig, Marius |
| Associated with | Stanford University, Cancer Biology Program. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Daniel Roberto Fuentes. |
|---|---|
| Note | Submitted to the Cancer Biology Program. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by Daniel Roberto Fuentes
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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