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Nucleosome Regulation of Transcription Factor Binding Kinetics: Implications for Gene Expression

Donovan, Benjamin Thomas
http://orcid.org/0000-0003-0177-799X
http://rave.ohiolink.edu/etdc/view?acc_num=osu1574774626880568

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Biophysics.
Compaction of genomic DNA into chromatin is an essential regulatory mechanism for eukaryotic gene expression. For example, nucleosomes drastically inhibit transcription factor (TF) binding, reducing occupancy approximately ~10,000-fold. This large decrease in TF occupancy is due to slower association and faster dissociation from binding sites within nucleosomes. Due to this effect, nucleosome positioning in promoter regions is tightly controlled. In budding yeast, General Regulatory Factors (GRFs) target binding sites in promoters and are essential for maintenance of proper nucleosome positioning. We show that the GRFs Reb1 and Cbf1 are Pioneer Factors because they bind nucleosome-embedded sites with similar affinities as to naked DNA. They bind by trapping nucleosomes in partially unwrapped conformations without histone eviction. Due to this reliance on nucleosome unwrapping, the binding rate is reduced by the probability of binding site exposure, about 100-fold. However, high affinity binding to nucleosomes is achieved by an equal reduction in their dissociation rates. Subsequent mutagenesis studies reveal that prolonged GRF dwell times on nucleosome-embedded binding sites requires only the DNA binding domain for both Reb1 and Cbf1. From these results, we propose that slowed dissociation from nucleosomes enables coordination with chromatin remodelers to establish and maintain nucleosome depleted regions. GRFs establish nucleosome-free regions in promoters which facilitate binding of transcriptional activators. In the GAL3 promoter, however, nucleosome mapping shows that the Gal4 binding site exists within a fragile nucleosome. In vitro single molecule measurements of Gal4 binding kinetics and in vivo single molecule measurements of transcriptional bursting reveals that the Gal4 dwell time sets the transcriptional burst size. Collectively, our data supports a model in which nucleosome positioning regulates RNA production via control of transcription factor binding kinetics.
Michael Poirier (Advisor)
Ralf Bundschuh (Committee Member)
Marcos Sotomayor (Committee Member)
Comert Kural (Committee Member)
236 p.
Biophysics
Chromatin; single molecule; nucleosomes; pioneer factors

Recommended Citations

Citations

  • Donovan, B. T. (2019). Nucleosome Regulation of Transcription Factor Binding Kinetics: Implications for Gene Expression [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574774626880568

    APA Style (7th edition)

  • Donovan, Benjamin. Nucleosome Regulation of Transcription Factor Binding Kinetics: Implications for Gene Expression. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1574774626880568.

    MLA Style (8th edition)

  • Donovan, Benjamin. "Nucleosome Regulation of Transcription Factor Binding Kinetics: Implications for Gene Expression." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574774626880568

    Chicago Manual of Style (17th edition)

osu1574774626880568
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This open access ETD is published by The Ohio State University and OhioLINK.