Purification of a promoter-specific and activator-dependent nucleosome disassembly factor
Abstract/Contents
- Abstract
- The disassembly of promoter nucleosomes is a requirement for the activation of eukaryotic genes. It renders the DNA accessible to the transcriptional machinery and primes it for the synthesis of messenger RNA by RNA Polymerase II. Promoter selection is achieved by the activator, a sequence-specific DNA binding protein that recognizes the promoter and recruits the nucleosome disassembly factor. The goals of this project were: 1. To establish an assay that recapitulates promoter-specific and activator-dependent nucleosome disassembly in vitro as it has been observed in vivo. 2. To identify the nucleosome disassembly factor responsible for this activity by purifying it from fractionated crude yeast extract and testing individual fractions using the aforementioned assay. As a model system, I chose the PHO5 promoter from yeast. Its activator, Pho4, is shuttled into the nucleus in response to phosphate starvation, where it binds to the promoter and triggers the disassembly of three nucleosomes. This process has been postulated to require an ATP-dependent nucleosome disassembly factor that is recruited via the activation domain of Pho4. Previous genetic and biochemical experiments have failed at revealing the identity of such a factor. I discovered that an activator-dependent nucleosome disassembly activity can be detected in vitro, that it depends on the activation domain of Pho4, and that it exhibits strong preference for the PHO5 promoter over its open-reading frame. As expected, the activity requires ATP. I developed a chromatographic fractionation protocol through which the activity was purified to a high degree of homogeneity. Mass spectrometry revealed the chromatin-remodeling complex Chd1 and the transcription elongation factor Sub1 to coelute with the activity. Purified Chd1 exhibited nucleosome disassembly activity and a chd1 deletion strain constitutively activated at PHO5 lacked expression of PHO5. These results pave the way for a further characterization of Chd1 in promoter-specific nucleosome disassembly and for the quest for additional stimulatory factors involved in this fundamental biochemical process.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Copyright date | 2011 |
| Publication date | 2009, c2011; 2009 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Ehrensberger, Andreas | |
|---|---|---|
| Associated with | Stanford University, Department of Biophysics. | |
| Primary advisor | Kornberg, Roger D | |
| Thesis advisor | Kornberg, Roger D | |
| Thesis advisor | Gozani, Or Pinchas | |
| Thesis advisor | Boeger, Hinrich | |
| Advisor | Gozani, Or Pinchas | |
| Advisor | Boeger, Hinrich |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Andreas Hasso Ehrensberger. |
|---|---|
| Note | Submitted to the Department of Biophysics. |
| Thesis | Ph.D. Stanford University 2010 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2010 by Andreas Ehrensberger
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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