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The Influence of DNA Sequence and Post Translational Modifications on Nucleosome Positioning and Stability

Mooney, Alex M.

Abstract Details

2012, Doctor of Philosophy, Ohio State University, Physics.

Eukaryotic DNA is organized into chromosomes by forming structural complexes with proteins. Naked DNA is wrapped around a hetero-octamer of histone proteins to form a nucleosome. Many nucleosomes on the same piece of DNA look like beads on a string. In a cell, the nucleosomes stack with each other to form a 30 nm fiber made of histone proteins and DNA. The 30 nm fiber is further folded onto itself with more protein until it has formed the familiar X-shaped chromosomes.

This research focuses on the nucleosome at the exclusion of the higher level structures. We are interested in the physical properties of the nucleosome, such as binding affinity and positioning, and methods of modifying those properties. We investigated the role that post-translational modifications (PTMs) can have in the formation and stability of nucleosomes. Traditionally, PTMs have been viewed as a way to recruit other proteins that can interact with a nucleosome, but we have shown modifications near DNA-protein contacts can alter the affinity of forming a nucleosome. Specifically, we tested H3-K115Ac/K122Ac (2KAc) synthetically prepared histones against recombinant histones with no modifications as well as mutated histones bearing mimics for that particular modification.

The formation and positioning of a nucleosome on a piece of DNA is highly dependent on the sequence of that DNA. Some sequences, such as long repeats of A's, have much lower affinity for nucleosomes than random DNA, while DNA with repeated, out of phase WW and SS dimers typically have very high affinity. Current in vitro nucleosome experiments typically use a synthetic sequence of DNA, called 601, that was discovered to have a higher affinity for nucleosomes than any known in vivo DNA sequence. Since the affinity of the synthetic sequence is so high, it has drawn criticism when used in experiments recently. We have used high throughput DNA sequencing data to discover a few natural DNA sequences from the Saccharomyces cerevisiae genome that position nucleosomes about as well as the synthetic DNA sequences. These will be useful for future experiments that require well-positioned nucleosomes.

We also used high throughput DNA sequencing to investigate a possible change in the global nucleosome positioning pattern across the Saccharomyces cerevisiae genome. We have found highly statistically significant differences in the distribution of nucleosomes when they were formed with unmodified histone octamer versus when they were modified with the same 2KAc PTMs we studied earlier. These findings are still in an early stage, but we present the evidence to date of nucleosome repositioning due to dyad modifications.

Michael Poirier, PhD (Advisor)
Ralf Bundschuh, PhD (Committee Member)
David Stroud, PhD (Committee Member)
Comert Kural, PhD (Committee Member)
Jeffery Parvin, PhD (Committee Member)
141 p.

Recommended Citations

Citations

  • Mooney, A. M. (2012). The Influence of DNA Sequence and Post Translational Modifications on Nucleosome Positioning and Stability [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354733493

    APA Style (7th edition)

  • Mooney, Alex. The Influence of DNA Sequence and Post Translational Modifications on Nucleosome Positioning and Stability. 2012. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1354733493.

    MLA Style (8th edition)

  • Mooney, Alex. "The Influence of DNA Sequence and Post Translational Modifications on Nucleosome Positioning and Stability." Doctoral dissertation, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354733493

    Chicago Manual of Style (17th edition)