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Reading the Epigenetic State of Chromatin Alters its Accessibility

Gibson, Matthew D
http://rave.ohiolink.edu/etdc/view?acc_num=osu1480534756664384

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Physics.
The eukaryotic genome is organized into a structural polymer called chromatin. Ultimately, all access to genetic information is regulated by chromatin including access required for DNA replication, transcription, and repair. The basic repeating unit of chromatin is the nucleosome which is comprised of ~147 bp of DNA tightly wrapped around a protein histone octamer core. The histone octamer is made up of eight proteins: two each of histones H2A, H2B, H3, and H4. Many mechanisms exist to regulate access to DNA but one of pivotal importance is the creation of unique nucleosomes through i) integration of histone variants and ii) deposition of post translational modifications (PTMs). These modifications help comprise the epigenome of a cell. Classically, the two mechanisms by which they function have been through a direct regulation of nucleosome dynamics, or through third party proteins which are able to recognize the variants or PTMs and facilitate work. The library of potential PTMs therefore forms a sort of histone code which regulates access to DNA. This thesis investigates the intersection of these mechanisms to determine whether the act of recognizing epigenetic information alters DNA accessibility. The primary method used to determine changes in DNA accessibility is though observing the effective binding affinity of a transcription factor to its target site buried within a recombinantly prepared nucleosome which has been modified to carry a PTM and to report on its wrapping state. We find different regulation depending both on the PTM we investigate and the specific PTM-binding protein. We first investigate the H3K36me3-binding protein PHF1 and find that while the PTM it recognizes, H3K36me3, does not alter DNA accessibility, the binding of its recognition domain and N-terminal domain can illicit a change of DNA accessibilty of 8 ± 2-fold. This means that 8 times less DNA binding protein is required to occupy its target site if the nucleosome is bound by PHF1. Second, we move to another PTM-binding protein, LEDGF/P75. Though LEDGF/P75 recognizes the same PTM as PHF1, we find that rather than increasing accessibility, LEDGF/P75 decreases accessibility to DNA. Additionally, we find that LEDGF/P75 prefers binding nucleosomes which simultaneously contain the histone variant H3.3 and its target PTM over those containing canonical H3.2 and the PTM. Finally, we shift from H3K36me3, a mark correlated to active transcription, to H3K9me3, which is correlated with compact, silent chromatin. The PTM-binding protein we investigate is Swi6 which, upon binding, initially increases DNA accessibility to nucleosomes by a factor of 2 ± 0.3. We further find that Swi6 maintains a roughly constant ratio of accessibilty between modified and unmodified nucleosomes of 3.2 ± 0.4. The findings presented here form a foundation for the study of the direct impact of the binding of PTM-recognition proteins. Future work will establish mechanistic insight into the alteration of DNA access within chromatin by PTM binding.
Michael Poirier, PhD (Advisor)
Ralf Bundschuh, PhD (Committee Member)
Comert Kural, PhD (Committee Member)
Fengyuan Yang, PhD (Committee Member)
Michael Barton, PhD (Committee Member)
233 p.
Biochemistry; Biology; Biophysics; Molecular Biology; Physics
Chromatin; Nucleosome; Histone; DNA Accessibility; DNA Regulation; Epigenetics; Epigenetic Regulation; Post Translational Modification; PTM; Histone Variant; PHF1; Swi6; HP1; LEDGF; P75; Transcription Factor; LexA; Single Molecule; FRET; Ligand Binding

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Citations

  • Gibson, M. D. (2016). Reading the Epigenetic State of Chromatin Alters its Accessibility [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480534756664384

    APA Style (7th edition)

  • Gibson, Matthew. Reading the Epigenetic State of Chromatin Alters its Accessibility. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1480534756664384.

    MLA Style (8th edition)

  • Gibson, Matthew. "Reading the Epigenetic State of Chromatin Alters its Accessibility." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480534756664384

    Chicago Manual of Style (17th edition)

osu1480534756664384
367
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This open access ETD is published by The Ohio State University and OhioLINK.