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

Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome

Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative <i>in vitro</i> studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin

The Effects of Histone H2B Ubiquitylations on the Nucleosome Structure and Internucleosomal Interactions

Eukaryotic gene compaction takes place at multiple levels to package DNA to chromatin and chromosomes. Two of the most fundamental levels of DNA packaging are at the nucleosome and dinucleosome stacks. The nucleosome is the basic gene-packing unit and is composed of DNA wrapped around a histone core. Nucleosomes stack with one another for further compaction of DNA. The first stacking step leads to dinucleosome formation, which is driven by internucleosomal interactions between various parts of two nucleosomes. Histone proteins are rich targets for post-translational modifications, some of which affect the structure of the nucleosome and the interactions between nucleosomes. These effects are often implicated in the regulation of various genomic transactions. In particular, histone H2B ubiquitylation has been associated with facilitated transcription and hexasome formation. Here, we employed semi-synthetically ubiquitylated histone H2B and single-molecule FRET to investigate the effects of H2B ubiquitylations at lysine 34 (H2BK34) and lysine 120 (H2BK120) on the structure of the nucleosome and the interactions between two nucleosomes. Our results suggest that H2BK34 ubiquitylation widens the DNA gyre gap in the nucleosome and stabilizes long- and short-range internucleosomal interactions while H2BK120 ubiquitylation does not affect the nucleosome structure or internucleosomal interactions. These results suggest potential roles for H2B ubiquitylations in facilitated transcription and hexasome formation while maintaining the structural integrity of chromatin

Gene Expression Profiles of Human Adipose Tissue-Derived Mesenchymal Stem Cells Are Modified by Cell Culture Density

<div><p>Previous studies conducted cell expansion <i>ex vivo</i> using low initial plating densities for optimal expansion and subsequent differentiation of mesenchymal stem cells (MSCs). However, MSC populations are heterogeneous and culture conditions can affect the characteristics of MSCs. In this study, differences in gene expression profiles of adipose tissue (AT)-derived MSCs were examined after harvesting cells cultured at different densities. AT-MSCs from three different donors were plated at a density of 200 or 5,000 cells/cm². After 7 days in culture, detailed gene expression profiles were investigated using a DNA chip microarray, and subsequently validated using a reverse transcription polymerase chain reaction (RT-PCR) analysis. Gene expression profiles were influenced primarily by the level of cell confluence at harvest. In MSCs harvested at ∼90% confluence, 177 genes were up-regulated and 102 genes down-regulated relative to cells harvested at ∼50% confluence (<i>P</i><0.05, FC>2). Proliferation-related genes were highly expressed in MSCs harvested at low density, while genes that were highly expressed in MSCs harvested at high density (∼90% confluent) were linked to immunity and defense, cell communication, signal transduction and cell motility. Several cytokine, chemokine and growth factor genes involved in immunosuppression, migration, and reconstitution of damaged tissues were up-regulated in MSCs harvested at high density compared with MSCs harvested at low density. These results imply that cell density at harvest is a critical factor for modulating the specific gene-expression patterns of heterogeneous MSCs.</p></div

Phase-contrast micrograph and cell density of AT-MSCs from three different donors in CC1 or CC2.

<p>(<b>A</b>) Morphological appearance of AT-MSC donors 7 days after plating at 200 cells/cm² (CC1) or 5,000 cells/cm² (CC2). All cells exhibited a spindle shaped or fibroblastic morphology. (<b>B</b>) The number of cell divisions and (<b>C</b>) total cell numbers at the time of harvest of MSCs cultured under different conditions. Data are the mean ± SD from three separate experiments.</p

RT-PCR analysis of differentially expressed cytokine, chemokine and proliferation-associated genes in AT-MSC from different donors and different cell densities.

<p>The expression profile of selected genes from the microarray data was validated by semi-quantitative RT-PCR using independent samples harvested 7days after plating at different cell densities as distinct from that for microarray analysis. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to GAPDH mRNA. Band intensity was normalized against that of GAPDH mRNA. Semi-quantitative RT-PCR analysis was independently performed using different MSC samples but the samples for microarray analysis. CC1, cultures plated with an initial cell density of 200 cells/cm² and a culture duration of 7 days; CC2, cultures plated with an initial cell density of 5,000 cells/cm² and a culture duration of 7 days.</p

Differentially expressed cell proliferation-associated genes in AT-MSCs from three different donors, cultured to low or high density, as determined by microarray analysis.

<p>Viable second-passage AT-MSCs plated at 200 cells/cm² (CC1 MSCs) or 5,000 cells/cm² (CC2 MSCs) were incubated for 7 days, by which time they reached ∼50% or ∼90% confluence, respectively. After harvesting, mRNA from three donor pooled samples of AT-MSCs was used in the microarray analysis. Microarray data were filtered by applying two criteria for significance, P<0.05 and FC>2 between culture conditions.</p

Hierarchical cluster analysis of differentially expressed genes in AT-MSCs from three different donors in CC1 or CC2.

<p>The microarray data for 47,323 genes were filtered by applying two criteria for significance, <i>P</i><0.05 and fold change (FC)>2, between the two cell densities (CC1 and CC2) at harvest for each MSC donor. (<b>A</b>) The selected data represented by hierarchical clustering of the normalized Ct of 279 genes on MSCs using individual samples (177 with increased expression, 102 with decreased expression). Each row represents a single gene, while each column represents the gene expression levels for a cell culture. The color coded gene expression levels range from red for the highest level of expression to green for the lowest. (<b>B</b>) Hierarchical cluster analysis of 17 differentially expressed cytokine, chemokine and growth factor genes. (<b>C</b>) Hierarchical cluster analysis of 33 differentially expressed proliferation-associated genes. CC1, cultures plated with an initial cell density of 200 cells/cm² and a culture duration of 7 days; CC2, cultures plated with an initial cell density of 5,000 cells/cm² and a culture duration of 7 days.</p