Introduction

Core histones wrapped around by DNA make nucleosomes, the fundamental packing units of the eukaryotic genome [1]. Each nucleosome core particle is composed of 145–147 base pair (bp) DNA and two copies of four core histones (H2A, H2B, H3 and H4) that are highly conserved throughout eukaryotes. Nucleosomal DNA bends extensively to form a left-handed superhelix with 1.65 turns around an octameric histone core which comprises a (H3–H4)2 tetramer and two H2A–H2B dimers [2]. Multiple nucleosome core particles joined by 10–90 bp linker DNA, linker histones and some other proteins form chromatin and eventually the chromosome. Electrostatic interactions between core histones and DNA are the main driving force of in vitro nucleosome reconstitution using salt dialysis. In a salt dialysis reconstitution method, the initial 2 M NaCl concentration of a sample solution with core histones and DNA is gradually decreased to 50–150 mM [3]. This method is based on the fact that core histones interact strongly with DNA at a physiological salt concentration while they are stable at a 2 M NaCl concentration without forming contacts with DNA. This reconstitution method suggests that the DNA/histone interactions are stabilized largely by hydrogen bonds and electrostatic interactions between the DNA backbone and the basic residues of histones (R or K) or the helix dipoles of histone-folds [1]. These interactions occur approximately every 10 bp at the minor grooves of nucleosomal DNA [4].

As most of the genes in a cell are wrapped tightly in nucleosomes that are further folded into chromatin, the molecular machines involved in various genomic transactions such as transcription, replication, repair, and recombination need the chromatin and/or nucleosome structure altered in order to access the genetic information. Mechanisms to switch between permissive and repressive states of chromatin conformation by various chromatin modifying and remodeling enzymes have been addressed in detail using various biochemical, molecular biology, epigenetic and single molecule techniques [5], [6], [7], [8], [9], [10], [11], [12], [13], [14].

Core histones share a highly conserved tertiary structure despite a low homology in their sequences. The structure represented by the two-fold repetition of a helix/loop/helix configuration with a high degree of helicity (~ 75%) is the molecular basis for the H2A/H2B and H3/H4 heterodimer formation via a handshake pairing [15]. A four-helix bundle is formed by two major helices and two small helices of two H3 molecules in a nucleosome. In the same way, H2B and H4 molecules also form a four-helix bundle. These helix bundles are the major motives of the octameric structure of a histone core. The residual N-terminal tails that account for ~ 28% of an octamer core are highly basic due to the abundant lysine and arginine residues and commonly hypothesized to be mostly unstructured [16]. Notably, the N-terminal tails of histones serve as major targets for several posttranslational modifications (PTM) such as acetylation, methylation, phosphorylation, ubiquitylation, and sumoylation that are linked to various gene regulatory activities [17], [18]. The N-terminal tails of histones may be in contact with DNA and other parts of histones in intra- and inter-nucleosomal contexts and consequently contribute to the regulation of the structures of nucleosomes and their packaging in chromatin [19], [20], [21].

Nucleosome core particles containing various DNA sequences, including a 146 bp fragment derived from the human X-chromosome α‐satellite DNA [1] and the Selex 601 sequence [22], [23], have been studied with crystallography [24]. A 146 bp fragment derived from a 5s RNA gene has also been used to reconstitute a nucleosome core particle, which was suggested to have two major translational positions [25], [26]. The Selex 601 sequence, which was selected from a large random DNA pool by a competition assay, exhibited a high level of affinity to histones with a strong 10 bp periodicity of the TA dinucleotide sequence that resulted in a highly homogeneous nucleosome position [23].

Fluorescence resonance energy transfer (FRET) is non-radiative energy transfer from a donor fluorophore to an acceptor fluorophore [27], [28]. The efficiency of FRET is a function of the distance between the two fluorophores. When the acceptor emission can be spectrally well separated from the donor emission, their intensities can be measured separately and the FRET efficiency can be easily calculated with the following equation:E=IA/IA+IDΦA/ΦDwhere IA and ID are the fluorescence intensities for the acceptor and the donor, respectively, and ΦA and ΦD are the fluorescence quantum yields of the acceptor and the donor, respectively.

FRET can be modeled as an induced dipole–dipole interaction between a donor and an acceptor. The transfer efficiency as a function of spectral overlap and spatial arrangements of the two fluorophores can be approximated as following.E=1/1+r/Ro6andRo6=2.8×10−28κ2ΦD∫IDλεAλλ4dλ,where r is the distance between the fluorophores, Ro is the Förster distance, κ2 is the orientation factor, ΦD is the donor fluorescence quantum yield, ID is the donor fluorescence emission, and εA is the acceptor absorption.

Combining the above two equations yieldsr6=ID/ΦD/IA/ΦARo6.

Based on this relationship, one can obtain the distance between the two fluorophores from their fluorescence intensities. When the intensities are measured from individual fluorophores in a time resolved manner, which is referred to as single molecule FRET or smFRET, one can follow the time trajectory of the distance between two fluorophores undergoing FRET.

To calculate the distance between two fluorophores from their FRET efficiency, Ro should be known or experimentally determined. Ro contains many known factors for commercially available fluorophores such as quantum yields and spectral overlap. It also contains the orientation factor κ2 that is 2/3 when the two dipoles are freely rotating. Restriction of fluorophore rotation requires κ2, specific to a given system, to be obtained experimentally or by modeling. For internal comparison of a distance in two different cases, one may avoid κ2 measurement by designing a case-specific approach as we previously demonstrated [12], [13]. Utilizing fluorescence anisotropy measurements, fixed fluorophores can be useful to evaluate the structural flexibility of a molecule on a ns timescale or to monitor the changes in the spatial arrangements of two points where the fluorophores are labeled [14].

Single molecule methods are currently being widely employed in studies of dynamic biological macromolecules. Single molecule approaches are particularly useful when the dynamic motion of the entity is complex or the change of interest is too small to be reliably resolved in an ensemble. By employing smFRET measurements we have reported changes, both in structures and structural dynamics of nucleosomes upon histone acetylation and DNA methylation, otherwise buried in noise in ensemble measurements [12], [13], [14].

It has long been hypothesized that some chromatin modifications may directly modulate the structure of nucleosomes and chromatin. In particular, histone acetylation and DNA methylation are globally conserved modifications and would have a high chance of affecting the physical properties of nucleosomes such as the structure and structural dynamics. We found that this hypothesis is valid based on our smFRET and fluorescence anisotropy measurements on various nucleosomes. In this review, we will summarize our findings in the effects of histone acetylation and DNA methylation on the structure and dynamics of nucleosomes addressed from single molecule approaches [12], [13], [14].

Section snippets

Effects of histone acetylation on the structure of nucleosomes and internucleosomal interactions

Core histone tails contain lysine residues that serve as targets for various histone acetyltransferases, which transfer an acetyl group of acetyl-coenzyme A (Ac-CoA) to the ε-amine group of a lysine residue [29], [30]. A long-standing hypothesis for histone acetylation affecting chromatin structure is based on charge neutralization of the lysine residues upon acetylation. The charge neutralization would reduce the affinity of lysine to DNA, resulting in an elevated level of DNA accessibility.

Effects of DNA methylation on the structure of nucleosomes

DNA methylation is associated with repressive chromatin states. It is carried out by a DNA methyltransferase which transfers a methyl group from S-adenosyl methionine (SAM) to the C5 position of cytosine in a CpG dinucleotide context in most cases [51]. Approximately 4% of the genomic DNA is hypermethylated primarily in the form of 5-methylcytosines in CpG dinucleotides [52]. CpG dinucleotides in vertebrate DNA appear at a frequency lower than the statistically expected value [53]. On the other

Conclusions

Recent advancement in identifying chromatin-binding proteins has shed light on how genes are regulated by the catalytic activities of these proteins and their interactions with chromatin. However, we still lack a basic biophysical understanding of how these proteins contribute to the regulation of the structure and structural dynamics of chromatin, which are another critical aspect of chromatin dynamics in cells. The new insights brought by recent studies in our lab demonstrate the richness of

Acknowledgements

Our research was funded by an NIH grant (GM097286), a Searle Scholar award and a Henry and Camille Dreyfus New Faculty Award to T.L.

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