Regulation of nucleosome dynamics

Ngo, Thuy

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Description

Title

Regulation of nucleosome dynamics

Author(s)

Ngo, Thuy

Issue Date

2015-01-21

Director of Research (if dissertation) or Advisor (if thesis)

Ha, Taekjip

Doctoral Committee Chair(s)

Ha, Taekjip

Committee Member(s)

• Belmont, Andrew S.
• Chemla, Yann
• Aksimentiev, Aleksei

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Computnl Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• nucleosomes
• unwrapping
• nucleosome dynamics
• asymmetry
• DNA sequence
• DNA flexibility
• DNA modifications
• optical tweezers
• fluorescence resonance energy transfer (FRET)
• single-molecule
• DNA cyclization
• gaping
• DNA methylation (5-mC)
• 5-Hydroxymethylcytosine (5-hmC)
• 5-formylcytosine (5-fC)
• 5-carboxylcytosine (5-caC)
• mismatch

Abstract

In eukaryotic cells, the genome is packed into fundamental units called nucleosomes, where 147 base pairs of DNA are wrapped around a protein core. Stable packing of DNA in nucleosomes imposes a barrier for accessibility of genetic code on DNA for replication, transcription, and repair. The dynamics of nucleosomal DNA provide a mean for gene regulation by genomic sequence and epigenetic modifications. Understanding the physical basis of how sequence and epigenetic modifications of DNA affect nucleosome dynamics and nucleosomal DNA exposure will help elucidate how genomic and epigenetic modifications regulate cellular functions, cell differentiation and cancer development. This motivated us to investigate local conformational dynamics of the nucleosome under tension or in the relaxed state and its modulation by DNA sequence and modifications. We achieved the goals by utilizing single-molecule force fluorescence spectroscopy, which allows monitoring dynamics of nucleosome at a define locality, and single-molecule DNA cyclization measurements, which enables determining of correlation of nucleosome dynamics with DNA flexibility. Chapter 2 shows details of sample preparation and methods used in studies of this dissertation. We made three profound discoveries: (1) the nucleosome unwraps directionally under force; (2) DNA flexibility is the basic physical property that controls nucleosome stability and nucleosomal DNA accessibility; and (3) two nucleosomal DNA ends are orchestrated such that the opening of one end helps stabilize the other end, providing a mechanism to amplify even a small difference in flexibility to a large mechanical asymmetry. These results are presented in Chapter 3. In Chapter 4 and 5, we present results to further demonstrate the correlation between DNA flexibility and unwrapping force by varying DNA modifications such as DNA mismatches, 5-methylcytosine and 5-formylcytosine. DNA methylation (5-methylcytosine) decreases DNA flexibility and reduces nucleosome mechanical stability while DNA mismatches and 5-formylcytosine have opposite effects. Our results suggest a completely new mechanism through which DNA sequence and epigenetic marks on DNA may be utilized to regulate gene expression by controlling nucleosome accessibility for replication, transcription, repair and remodeling. Finally, we identified slow spontaneous local gaping of nucleosomes under physiological conditions. Gaping modes switch along the direction normal to the DNA plane at minutes (1-10 minutes) time scale. The existence of nucleosome in different gaping modes may underlie the heterogeneous enzymatic reactions on chromatin substrates and the formation of multiple compression forms of chromatin fibers. These results are detailed in Chapter 6.

Graduation Semester

2014-12

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Copyright and License Information

Copyright 2014 Thuy T. M. Ngo

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