Role of histone tails in structural stability of the nucleosome

Histone tails play an important role in nucleosome structure and dynamics. Here we investigate the effect of truncation of histone tails H3, H4, H2A and H2B on nucleosome structure with 100 ns all-atom molecular dynamics simulations. Tail domains of H3 and H2B show propensity of α-helics formation during the intact nucleosome simulation. On truncation of H4 or H2B tails no structural change occurs in histones. However, H3 or H2A tail truncation results in structural alterations in the histone core domain, and in both the cases the structural change occurs in the H2Aα3 domain. We also find that the contacts between the histone H2A C terminal docking domain and surrounding residues are destabilized upon H3 tail truncation. The relation between the present observations and corresponding experiments is discussed.     

Histone tail network and modulation in a nucleosome

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A/H2B heterodimers and one histone (H3/H4)₂ tetramer, wrapped around by ∼146-bp core DNA and linker DNA. Flexible histone tails sticking out from the core undergo posttranslational modifications that are responsible for various epigenetic functions. Recently, the functional dynamics of histone tails and their modulation within the nucleosome and nucleosomal complexes have been investigated by integrating NMR, molecular dynamics simulations, and cryo-electron microscopy approaches. In particular, recent NMR studies have revealed correlations in the structures of histone N-terminal tails between H2A and H2B, as well as between H3 and H4 depending on linker DNA, suggesting that histone tail networks exist even within the nucleosome.     

Effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and the interactions between two nucleosomes

We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (～1 s) states. The formation of dinucleosomes is strongly Mg(2+)-dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ～2 nm face-to-face and rotated by 34° with respect to each other.     

The effect of epigenetic modifications on the secondary structures and possible binding positions of the N-terminal tail of histone H3 in the nucleosome: a computational study

The roles of histone tails as substrates for reversible chemical modifications and dynamic cognate surfaces for the binding of regulatory proteins are well established. Despite these crucial roles, experimentally derived knowledge of the structure and possible binding sites of histone tails in chromatin is limited. In this study, we utilized molecular dynamics of isolated histone H3 N-terminal peptides to investigate its structure as a function of post-translational modifications that are known to be associated with defined chromatin states. We observed a structural preference for α-helices in isoforms associated with an inactive chromatin state, while isoforms associated with active chromatin states lacked α-helical content. The physicochemical effect of the post-translational modifications was highlighted by the interaction of arginine side-chains with the phosphorylated serine residues in the inactive isoform. We also showed that the isoforms exhibit different tail lengths, and, using molecular docking of the first 15 N-terminal residues of an H3 isoform, identified potential binding sites between the superhelical gyres on the octamer surface, close to the site of DNA entry/exit in the nucleosome. We discuss the possible functional role of the binding of the H3 tail within the nucleosome on both nucleosome and chromatin structure and stability.     

Histone tails cooperate to control the breathing of genomic nucleosomes

Genomic DNA is packaged in chromatin, a dynamic fiber variable in size and compaction. In chromatin, repeating nucleosome units wrap 145–147 DNA basepairs around histone proteins. Genetic and epigenetic regulation of genes relies on structural transitions in chromatin which are driven by intra- and inter-nucleosome dynamics and modulated by chemical modifications of the unstructured terminal tails of histones. Here we demonstrate how the interplay between histone H3 and H2A tails control ample nucleosome breathing motions. We monitored large openings of two genomic nucleosomes, and only moderate breathing of an engineered nucleosome in atomistic molecular simulations amounting to 24 μs. Transitions between open and closed nucleosome conformations were mediated by the displacement and changes in compaction of the two histone tails. These motions involved changes in the DNA interaction profiles of clusters of epigenetic regulatory aminoacids in the tails. Removing the histone tails resulted in a large increase of the amplitude of nucleosome breathing but did not change the sequence dependent pattern of the motions. Histone tail modulated nucleosome breathing is a key mechanism of chromatin dynamics with important implications for epigenetic regulation.     

Dissecting DNA-histone interactions in the nucleosome by molecular dynamics simulations of DNA unwrapping

The nucleosome complex of DNA wrapped around a histone protein octamer organizes the genome of eukaryotes and regulates the access of protein factors to the DNA. We performed molecular dynamics simulations of the nucleosome in explicit water to study the dynamics of its histone-DNA interactions. A high-resolution histone-DNA interaction map was derived that revealed a five-nucleotide periodicity, in which the two DNA strands of the double helix made alternating contacts. On the 100-ns timescale, the histone tails mostly maintained their initial positions relative to the DNA, and the spontaneous unwrapping of DNA was limited to 1–2 basepairs. In steered molecular dynamics simulations, external forces were applied to the linker DNA to investigate the unwrapping pathway of the nucleosomal DNA. In comparison with a nucleosome without the unstructured N-terminal histone tails, the following findings were obtained: 1), Two main barriers during unwrapping were identified at DNA position ±70 and ±45 basepairs relative to the central DNA basepair at the dyad axis. 2), DNA interactions of the histone H3 N-terminus and the histone H2A C-terminus opposed the initiation of unwrapping. 3), The N-terminal tails of H2A, H2B, and H4 counteracted the unwrapping process at later stages and were essential determinants of nucleosome dynamics. Our detailed analysis of DNA-histone interactions revealed molecular mechanisms for modulating access to nucleosomal DNA via conformational rearrangements of its structure.     

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.     

Multiscale modeling of nucleosome dynamics

Nucleosomes form the fundamental building blocks of chromatin. Subtle modifications of the constituent histone tails mediate chromatin stability and regulate gene expression. For this reason, it is important to understand structural dynamics of nucleosomes at atomic levels. We report a novel multiscale model of the fundamental chromatin unit, a nucleosome, using a simplified model for rapid discrete molecular dynamics simulations and an all-atom model for detailed structural investigation. Using a simplified structural model, we perform equilibrium simulations of a single nucleosome at various temperatures. We further reconstruct all-atom nucleosome structures from simulation trajectories. We find that histone tails bind to nucleosomal DNA via strong salt-bridge interactions over a wide range of temperatures, suggesting a mechanism of chromatin structural organization whereby histone tails regulate inter- and intranucleosomal assemblies via binding with nucleosomal DNA. We identify specific regions of the histone core H2A/H2B-H4/H3-H3/H4-H2B/H2A, termed “cold sites”, which retain a significant fraction of contacts with adjoining residues throughout the simulation, indicating their functional role in nucleosome organization. Cold sites are clustered around H3-H3, H2A-H4 and H4-H2A interhistone interfaces, indicating the necessity of these contacts for nucleosome stability. Essential dynamics analysis of simulation trajectories shows that bending across the H3-H3 is a prominent mode of intranucleosomal dynamics. We postulate that effects of salts on mononucleosomes can be modeled in discrete molecular dynamics by modulating histone-DNA interaction potentials. Local fluctuations in nucleosomal DNA vary significantly along the DNA sequence, suggesting that only a fraction of histone-DNA contacts make strong interactions dominating mononucleosomal dynamics. Our findings suggest that histone tails have a direct functional role in stabilizing higher-order chromatin structure, mediated by salt-bridge interactions with adjacent DNA.     

Histone H4 K16Q mutation, an acetylation mimic, causes structural disorder of its N-terminal basic patch in the nucleosome

Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19).     

Structure and dynamics of nucleosomal DNA

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.     

Breaths,Twists, and Turns of Atomistic Nucleosomes

Gene regulation programs establish cellular identity and rely on dynamic changes in the structural packaging of genomic DNA. The DNA is packaged in chromatin, which is formed from arrays of nucleosomes displaying different degree of compaction and different lengths of inter-nucleosomal linker DNA. The nucleosome represents the repetitive unit of chromatin and is formed by wrapping 145–147 basepairs of DNA around an octamer of histone proteins. Each of the four histones is present twice and has a structured core and intrinsically disordered terminal tails. Chromatin dynamics are triggered by inter- and intra-nucleosome motions that are controlled by the DNA sequence, the interactions between the histone core and the DNA, and the conformations, positions, and DNA interactions of the histone tails. Understanding chromatin dynamics requires studying all these features at the highest possible resolution. For this, molecular dynamics simulations can be used as a powerful complement or alternative to experimental approaches, from which it is often very challenging to characterize the structural features and atomic interactions controlling nucleosome motions. Molecular dynamics simulations can be performed at different resolutions, by coarse graining the molecular system with varying levels of details. Here we review the successes and the remaining challenges of the application of atomic resolution simulations to study the structure and dynamics of nucleosomes and their complexes with interacting partners.     

Two Arginine Residues Suppress the Flexibility of Nucleosomal DNA in the Canonical Nucleosome Core

The dynamics of nucleosomes containing either canonical H3 or its centromere-specific variant CENP-A were investigated using molecular dynamics simulations. The simulations showed that the histone cores were structurally stable during simulation periods of 100 ns and 50 ns, while DNA was highly flexible at the entry and exit regions and partially dissociated from the histone core. In particular, approximately 20–25 bp of DNA at the entry and exit regions of the CENP-A nucleosome exhibited larger fluctuations than DNA at the entry and exit regions of the H3 nucleosome. Our detailed analysis clarified that this difference in dynamics was attributable to a difference in two basic amino acids in the αN helix; two arginine (Arg) residues in H3 were substituted by lysine (Lys) residues at the corresponding sites in CENP-A. The difference in the ability to form hydrogen bonds with DNA of these two residues regulated the flexibility of nucleosomal DNA at the entry and exit regions. Our exonuclease III assay consistently revealed that replacement of these two Arg residues in the H3 nucleosome by Lys enhanced endonuclease susceptibility, suggesting that the DNA ends of the CENP-A nucleosome are more flexible than those of the H3 nucleosome. This difference in the dynamics between the two types of nucleosomes may be important for forming higher order structures in different phases.     

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)₂ tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.     

Simulations of SIN mutations and histone variants in human nucleosomes reveal altered protein-DNA and core histone interactions

Alterations in the stability of a nucleosome exert predominant influence on chromatin structure and eukaryotic gene expression. In an attempt to investigate the mononucleosome stability using computational approaches, we have simulated the structure of a human mononucleosome and have compared their energies under the influence of core mutations, tail substitutions, variant histones, and orthologs. We observe that mutant nucleosomes carrying SIN (SWI Independent) mutations do not alter the overall nucleosomal structure but cause local structural changes leading to significant changes in energy and hence the stability. We observe that the nucleosome stability is altered by the substitution of only certain critical lysine residues on the H3 tails. Interestingly, the incorporation of variants H2A.Z and H3.3 lower nucleosome stability as evidenced by small energy changes. However, the substitution of histone orthologs did not alter structural stability. Our simulations to determine the nucleosome stability using energy trends emphasize the role of mutations, variants, and orthologs as determinants of chromatin structure at the nucleosome core particle level. The destabilization we observe on the human nucleosome with core mutations show similar trends of instability as validated experimentally in yeast.     

Simulations of SIN Mutations and Histone Variants in Human Nucleosomes Reveal Altered Protein-DNA and Core Histone Interactions

Abstract Alterations in the stability of a nucleosome exert predominant influence on chromatin structure and eukaryotic gene expression. In an attempt to investigate the mononucleosome stability using computational approaches, we have simulated the structure of a human mononucleosome and have compared their energies under the influence of core mutations, tail substitutions, variant histones, and orthologs. We observe that mutant nucleosomes carrying SIN (SWI Independent) mutations do not alter the overall nucleosomal structure but cause local structural changes leading to significant changes in energy and hence the stability. We observe that the nucleosome stability is altered by the substitution of only certain critical lysine residues on the H3 tails. Interestingly, the incorporation of variants H2A.Z and H3.3 lower nucleosome stability as evidenced by small energy changes. However, the substitution of histone orthologs did not alter structural stability. Our simulations to determine the nucleosome stability using energy trends emphasize the role of mutations, variants, and orthologs as determinants of chromatin structure at the nucleosome core particle level. The de-stabilization we observe on the human nucleosome with core mutations show similar trends of instability as validated experimentally in yeast.