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Nucleosome Regulation of Transcription Factor Binding Dynamics: a Single-molecule Study

Luo, Yi
http://rave.ohiolink.edu/etdc/view?acc_num=osu1449093157

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Biophysics.
Nucleosome is the most basic structural and functional unit of the eukaryotic genome. In each nucleosome, 147 base pairs of genomic DNA is wrapped 1.6 turns around a histone octamer protein core. The structural dynamics of nucleosome plays important role in the regulation of many gene-related cellular processes such as transcription activation, DNA replication and repair. This dissertation work focuses on the interplay between nucleosomal DNA unwrapping and transcription factor (TF) accessing the target site within the nucleosome. TFs are important regulatory proteins that bind their target sites on the genomic DNA to activate or repress transcription. The access of TF is blocked if a nucleosome is positioned on the target site, which occurs often in the genome. The nucleosomal DNA at the entry/exit region of the nucleosome undergoes rapid spontaneous unwrapping and rewrapping from the histone core under thermal fluctuation, allowing transient exposure of the target site for binding. We employed Fluorescence Resonance Energy Transfer (FRET) method to probe the conformation changes of nucleosome unwrapping and detect TF binding to the nucleosome-protected sites. Single-molecule Total-internal-reflection fluorescence (smTIRF) microscopy was used to track this dynamic process in real time. It was revealed in this study that nucleosomes regulate TF binding not only by reducing its association rate with limited site exposure, but also by accelerating the dissociation of TF from a nucleosomal site. The results showed that the overall TF dissociation rate is enhanced by a factor of 2-3 orders of magnitude. This enhanced dissociation appears to be a new general mechanism by which nucleosomes could regulate TF occupancy in the genome. Further investigations showed that this enhanced dissociation rate remains largely unchanged as the binding site is moved across a long stretch of DNA in the entry/exit region of nucleosome, and the TF occupancy at these sites is mainly regulated by the nucleosome unwrapping rate. Taken together, these studies provided detailed view about the influence of nucleosome dynamics on TF binding. We expanded the study to include linker histone H1 into this dynamic picture. H1 binds to the nucleosome and the linker DNA, further compacts the nucleosome array and is important in the organization of chromatin higher-order structures. Our studies using FRET and Protein-induced fluorescence enhancement showed that H1 reduces TF occupancy but does not completely block its access. The nucleosome can still unwrap with H1 bound at a much lower rate. Similar results have been observed with several H1 isoforms, suggesting that it is a general mechanism by which H1 regulates TF binding in the chromatin. We also used the smTIRF microscopy tool to characterize the dynamics of a DNA Origami Two-state (DOT) nanodevice. This device can be used as a sensor for the entropic depletion force caused by macromolecular crowding. We have shown that the DOT device is sensitive to 10 femtonewton forces and the dynamic range can be fine tuned by simple modifications in the structure design.
Michael Poirier, Dr. (Advisor)
Ralf Bundschuh, Dr. (Committee Member)
Carlos Castro, Dr. (Committee Member)
Richard Swenson, Dr. (Committee Member)
309 p.
Biochemistry; Biology; Biomechanics; Biophysics; Molecular Biology; Physics
nucleosome dynamics; transcription factor; single-molecule; TIRF; FRET; DNA origami;

Recommended Citations

Citations

  • Luo, Y. (2015). Nucleosome Regulation of Transcription Factor Binding Dynamics: a Single-molecule Study [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449093157

    APA Style (7th edition)

  • Luo, Yi. Nucleosome Regulation of Transcription Factor Binding Dynamics: a Single-molecule Study. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1449093157.

    MLA Style (8th edition)

  • Luo, Yi. "Nucleosome Regulation of Transcription Factor Binding Dynamics: a Single-molecule Study." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449093157

    Chicago Manual of Style (17th edition)

osu1449093157
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This open access ETD is published by The Ohio State University and OhioLINK.