Nucleosome dynamics studied with single-molecule methods

Open Access
- Author:
- Das, Subhra Kanti
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 17, 2024
- Committee Members:
- Paul Cremer, Major Field Member
Mark Hedglin, Major Field Member
Song Tan, Outside Unit & Field Member
Tae-Hee Lee, Chair & Dissertation Advisor
Philip Bevilacqua, Program Head/Chair - Keywords:
- Single-molecule fluorescence resonance energy transfer (smFRET)
Nucleosome dynamics
Spontaneous histone exchange
Post-translational modifications (PTMs)
Linker histone H1
H1 CTD dynamics
Total internal-reflection microscopy (TIR)
Site-specific DNA labeling
Site-specific protein labeling
CpG methylation - Abstract:
- In eukaryotes, the nucleosome is the primary gene-packing unit structure. The dynamic site exposure regulates chromatin accessibility, while the strong DNA-histone interactions impose physical barriers to DNA binding and processing. During the first few chapters of this dissertation, the fate of site exposure resulting in spontaneous histone dimer exchange between nucleosomes has been explained. Three-color single-molecule FRET (smFRET) investigations reveal that the timescale of spontaneous histone exchange is a few tens of seconds at a physiological nucleosome concentration. Histone exchange rates are elevated at a higher monovalent salt concentration, with histone acetylation, and in the presence of histone chaperone Nap1. Exchange rates are unchanged at a higher temperature and decreased upon DNA CpG methylation. Histone diffusion in a compact chromatin context can modulate local concentrations of post-translational histone modifications and histone variants. In the later chapters of this dissertation, conformational dynamics within the H1 C-terminal domain (CTD) and the role of histone H3-tail in CTD condensation dynamics have been emphasized. The disordered H1 CTD undergoes extensive condensation upon binding to nucleosomes. H1 stabilizes nucleosomes and higher-order chromatin structures, but its interactions in chromatin are poorly defined. Using smFRET, we report that about half of the H1 CTDs in H1-nucleosome complexes exhibit exchange between multiple defined FRET structures. Crosslinking study identifies localized contacts between linker DNA (~ first 25 bp) and H1 CTD (residues 1-30) and reports that two regions in the CTD contribute to the H1-dependent organization of linker DNA. Our results indicate that the nucleosome bound H1 CTD adopts loosely defined structures. We also report a suppressed condensation of nucleosome bound H1 CTD upon epigenetic acetylation within the H3 tail.