Solution Scattering and FRET Studies on Nucleosomes Reveal DNA Unwrapping Effects of H3 and H4 Tail Removal

Using a combination of small-angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) measurements we have determined the role of the H3 and H4 histone tails, independently, in stabilizing the nucleosome DNA terminal ends from unwrapping from the nucleosome core. We have performed solution scattering experiments on recombinant wild-type, H3 and H4 tail-removed mutants and fit all scattering data with predictions from PDB models and compared these experiments to complementary DNA-end FRET experiments. Based on these combined SAXS and FRET studies, we find that while all nucleosomes exhibited DNA unwrapping, the extent of this unwrapping is increased for nucleosomes with the H3 tails removed but, surprisingly, decreased in nucleosomes with the H4 tails removed. Studies of salt concentration effects show a minimum amount of DNA unwrapping for all complexes around 50-100mM of monovalent ions. These data exhibit opposite roles for the positively-charged nucleosome tails, with the ability to decrease access (in the case of the H3 histone) or increase access (in the case of the H4 histone) to the DNA surrounding the nucleosome. In the range of salt concentrations studied (0-200mM KCl), the data point to the H4 tail-removed mutant at physiological (50-100mM) monovalent salt concentration as the mononucleosome with the least amount of DNA unwrapping.     

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.     

Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure—a FRET study

Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.     

Nucleosomes structure and dynamics: effect of CHAPS

Dynamics of nucleosomes and spontaneous unwrapping of DNA are fundamental property of the chromatin enabling access to nucleosomal DNA for regulatory proteins. Probing of such dynamics of nucleosomes performed by single molecule techniques revealed a large scale dynamics of nucleosomes including their spontaneous unwrapping. Dissociation of nucleosomes at low concentrations is a complicating issue for studies with single molecule techniques. In this paper, we tested the ability of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) to prevent dissociation of nucleosomes. The study was performed with mononucleosome system assembled with human histones H2A, H2B, H3 and H4 on the DNA substrate containing sequence 601 that provides the sequencespecific assembly of nucleosomes. We used Atomic Force Microscopy (AFM) to directly identify nucleosomes and analyze their structure at the nanometer level. These studies showed that in the presence of CHAPS at millimolar concentrations, nucleosomes, even at sub-nanomolar concentrations, remain intact over days compared to a complete dissociation of the same nucleosome sample over 10 min in the absence of CHAPS. Importantly, CHAPS does not change the conformation of nucleosomes as confirmed by the AFM analysis. Moreover, 16 µM CHAPS stabilizes nucleosomes in over one hour incubation in the solution containing as low as 0.4 nM in nucleosomes. The stability of nucleosomes is slightly reduced at physiological conditions (150 mM NaCl), although the nucleosomes dissociate rapidly at 300 mM NaCl. The sequence specificity of the nucleosome in the presence of CHAPS decreased suggesting that the histone core translocates along the DNA substrate utilizing sliding mechanism.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.     

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.     

Nucleosome structural dynamics and super spiral linking number paradox

The minireview presents recent results obtained in the experiments with DNA minicircles containing reconstituted nucleosomes. This system allows one to register and to characterize conformational dynamics of nucleosomes and of subnucleosomal particles containing histone tetramer (H3-H4)2. In particular, it has revealed an important role of the histone N-terminal tails in this dynamics. Solution of the linking number paradox and relevance of the results obtained to chromatin structural dynamics are discussed.     

Nucleosome Structural Dynamics and Linking Number Paradox

The minireview presents recent results obtained in the experiments with DNA minicircles containing reconstituted nucleosomes. This system allows one to register and to characterize the conformational dynamics of nucleosomes and of subnucleosomal particles containing histone tetramer (H3-H4)₂. In particular, it has revealed an important role of the histone N-terminal tails in this dynamics. Solution of the linking number paradox and the relevance of the results obtained to chromatin structural dynamics are discussed.     

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)₂, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.     

Dynamics of Forced Nucleosome Unraveling and Role of Nonuniform Histone-DNA Interactions

A coarse-grained model of the nucleosome is introduced to investigate the dynamics of force-induced unwrapping of DNA from histone octamers. In this model, the DNA is treated as a charged, discrete worm-like chain, and the octamer is treated as a rigid cylinder carrying a positively charged superhelical groove that accommodates 1.7 turns of DNA. The groove charges are parameterized to reproduce the nonuniform histone/DNA interaction free energy profile and the loading rate-dependent unwrapping forces, both obtained from single-molecule experiments. Brownian dynamics simulations of the model under constant loading conditions reveal that nucleosome unraveling occurs in three distinct stages. At small extensions, the flanking DNA exhibits rapid unwrapping-rewrapping (breathing) dynamics and the octamer flips ～180° and moves toward the pulling axis. At intermediate extensions, the outer turn of DNA unwraps gradually and the octamer swivels about the taut linkers and flips a further ～90° to orient its superhelical axis almost parallel to the pulling axis. At large extensions, a portion of the inner turn unwraps abruptly with a notable rip in the force-extension plot and a >90° flip of the octamer. The remaining inner turn unwraps reversibly to leave a small portion of DNA attached to the octamer despite extended pulling. Our simulations further reveal that the nonuniform histone/DNA interactions in canonical nucleosomes serve to: stabilize the inner turn against unraveling while enhancing the breathing dynamics of the nucleosome and prevent dissociation of the octamer from the DNA while facilitating its mobility along the DNA. Thus, the modulation of the histone/DNA interactions could constitute one possible mechanism for regulating the accessibility of the nucleosome-wound DNA sequences.     

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.     

Histone tails and the H3 alphaN helix regulate nucleosome mobility and stability

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 alphaN helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.     

Sequence-dependent nucleosome structural and dynamic polymorphism. Potential involvement of histone H2B N-terminal tail proximal domain

Relaxation of nucleosomes on an homologous series (pBR) of ca 350-370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by approximately 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a approximately 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance ( approximately 20 bp) on H3-tail interactions with the small groove at the nucleosome entry-exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them.     

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.     

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.     

Intra- and inter-nucleosome interactions of the core histone tail domains in higher-order chromatin structure

Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array–array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, effect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as inter-nucleosomal and inter-array interactions of the H3 and H4 N-terminal tails.     

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.     

CENP-A-containing nucleosomes: easier disassembly versus exclusive centromeric localization

CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-containing nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the alpha-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(+/-2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)2 tetramer was also more readily destabilized with heparin than the (H3-H4)2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, especially as is seen following CENP-A transient over-expression.