Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2A-H2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA

Lysine Acetylation Facilitates Spontaneous DNA Dynamics in the Nucleosome

The nucleosome, comprising a histone protein core wrapped around by DNA, is the fundamental packing unit of DNA in cells. Lysine acetylation at the histone core elevates DNA accessibility in the nucleosome, the mechanism of which remains largely unknown. By employing our recently developed hybrid single molecule approach, here we report how the structural dynamics of DNA in the nucleosome is altered upon acetylation at histone H3 lysine 56 (H3K56) that is critical for elevated DNA accessibility. Our results indicate that H3K56 acetylation facilitates the structural dynamics of the DNA at the nucleosome termini that spontaneously and repeatedly open and close on a ms time scale. The results support a molecular mechanism of histone acetylation in catalyzing DNA unpacking whose efficiency is ultimately limited by the spontaneous DNA dynamics at the nucleosome temini. This study provides the first and unique experimental evidence revealing a role of protein chemical modification in directly regulating the kinetic stability of the DNA packing unit

Single-Molecule Observation Reveals Spontaneous Protein Dynamics in the Nucleosome

Structural dynamics of a protein molecule is often critical to its function. Single-molecule methods provide efficient ways to investigate protein dynamics, although it is very challenging to achieve a millisecond or higher temporal resolution. Here we report spontaneous structural dynamics of the histone protein core in the nucleosome based on a single-molecule method that can reveal submillisecond dynamics by combining maximum likelihood estimation and fluorescence correlation spectroscopy. The nucleosome, comprising ∼147 bp DNA and an octameric histone protein core consisting of H2A, H2B, H3, and H4, is the fundamental packing unit of the eukaryotic genome. The nucleosome imposes a physical barrier that should be overcome during various DNA-templated processes. Structural fluctuation of the nucleosome in the histone core has been hypothesized to be required for nucleosome disassembly but has yet to be directly probed. Our results indicate that at 100 mM NaCl the histone H2A–H2B dimer dissociates from the histone core transiently once every 3.6 ± 0.6 ms and returns to its position within 2.0 ± 0.3 ms. We also found that the motion is facilitated upon H3K56 acetylation and inhibited upon replacing H2A with H2A.Z. These results provide the first direct examples of how a localized post-translational modification or an epigenetic variation affects the kinetic and thermodynamic stabilities of a macromolecular protein complex, which may directly contribute to its functions