Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.     

How does the histone code work?

Patterns of histone post-translational modifications correlate with distinct chromosomal states that regulate access to DNA, leading to the histone-code hypothesis. However, it is not clear how modification of flexible histone tails leads to changes in nucleosome dynamics and, thus, chromatin structure. The recent discovery that, like the flexible histone tails, the structured globular domain of the nucleosome core particle is also extensively modified adds a new and exciting dimension to the histone-code hypothesis, and calls for the re-examination of current models for the epigenetic regulation of chromatin structure. Here, we review these findings and other recent studies that suggest the structured globular domain of the nucleosome core particle plays a key role regulating chromatin dynamics.     

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.     

Computer modeling demonstrates that electrostatic attraction of nucleosomal DNA is mediated by histone tails

We conducted molecular dynamics computer simulations of charged histone tail-DNA interactions in systems mimicking nucleosome core particles (NCP) . In a coarse-grained model, the NCP is modeled as a negatively charged spherical particle with flexible polycationic histone tails attached to it in a dielectric continuum with explicit mobile counterions and added salt. The size, charge, and distribution of the tails relative to the core were built to mimick real NCP. In this way, we incorporate attractive ion-ion correlation effects due to fluctuations in the ion cloud and the attractive entropic and energetic tail-bridging effects. In agreement with experimental data, increase of monovalent salt content from salt-free to physiological concentration leads to the formation of NCP aggregates; likewise, in the presence of MgCl₂, the NCPs form condensed systems via histone-tail bridging and accumulation of counterions. More detailed mechanisms of the histone tail-DNA interactions and dynamics have been obtained from all-atom molecular dynamics simulations (including water), comprising three DNA 22-mers and 14 short fragments of the H4 histone tail (amino acids 5–12) carrying three positive charges on lysine⁺ interacting with DNA. We found correlation of the DNA-DNA distance with the presence and association of the histone tail between the DNA molecules.     

“Direct” and “Indirect” Effects of Histone Modifications: Modulation of Sterical Bulk as a Novel Source of Functionality

The chromatin-regulatory principles of histone post-translational modifications (PTMs) are discussed with a focus on the potential alterations in chromatin functional state due to steric and mechanical constraints imposed by bulky histone modifications such as ubiquitin and SUMO. In the classical view, PTMs operate as recruitment platforms for histone “readers,” and as determinants of chromatin array compaction. Alterations of histone charges by “small” chemical modifications (e.g., acetylation, phosphorylation) could regulate nucleosome spontaneous dynamics without globally affecting nucleosome structure. These fluctuations in nucleosome wrapping can be exploited by chromatin-processing machinery. In contrast, ubiquitin and SUMO are comparable in size to histones, and it seems logical that these PTMs could conflict with canonical nucleosome organization. An experimentally testable hypothesis that by adding sterical bulk these PTMs can robustly alter nucleosome primary structure is proposed. The model presented here stresses the diversity of mechanisms by which histone PTMs regulate chromatin dynamics, primary structure and, hence, functionality.     

Structure of the chromatin with long linker DNA

The method of velocity sedimentation have been used to investigate ionic-strength-induced compaction of sea urchin sperm chromatin characterized by extremely long linker DNA (100 b.p.). The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain have been studied in the range of ionic strength from 0.005 to 0.085. Analysis of these data indicates that such structural parameters of sea urchin sperm chromatin fibre as the diameter of the chain and the length of the chain per nucleosome are quite similar to those of chromatin with shorter linker DNA, but the DNA packing ratio is higher. The structure of sea urchin sperm oligonucleosomes agrees well with the model of three-dimensional zig-zag-shaped chain with linker DNA forming a loop. The possible role of alpha-helical regions of the C-terminal domain of sea urchin sperm histone H1 in the long linker DNA folding is discussed.     

Probing the effects of DNA-protein interactions on DNA hole transport: the N-terminal histone tails modulate the distribution of oxidative damage and chemical lesions in the nucleosome core particle

The ability of DNA to transport positive charges, or holes, over long distances is well-established, but the mechanistic details of how this process is influenced by packaging into DNA-protein complexes have not been fully delineated. In eukaryotes, genomic DNA is packaged into chromatin through its association with the core histone octamer to form the nucleosome core particle (NCP), a complex whose structure can be modulated through changes in the local environment and the histone proteins. Because (i) varying the salt concentration and removing the histone tails influence the structure of the NCP in known ways and (ii) previous studies have shown that DNA hole transport (HT) occurs in the nucleosome, we have used our previously described 601 sequence NCPs to test the hypothesis that DNA HT dynamics can be modulated by structural changes in a DNA-protein complex. We show that at low salt concentrations there is a sharp increase in long-range DNA HT efficiency in the NCP as compared to naked DNA. This enhancement of HT can be negated by either removal of the histone tails at low salt concentrations or disruption of the interaction of the packaged DNA and the histone tails by increasing the buffer's ionic strength. Association of the histone tails with 601 DNA at low salt concentrations shifts the guanine damage spectrum to favor lesions like 8-oxoguanine in the NCP, most likely through modulation of the rate of the reaction of the guanine radical cation with oxygen. These experimental results indicate that for most genomic DNA, the influence of DNA-protein interactions on DNA HT will depend strongly on the level of protection of the DNA nucleobases from oxygen. Further, these results suggest that the oxidative damage arising from DNA HT may vary in different genomic regions depending on the presence of either euchromatin or heterochromatin.     

Regulated nucleosome mobility and the histone code

Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA. Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface. We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics.     

组蛋白变体及组蛋白替换

组蛋白作为核小体的基本组分,是染色质的结构和功能必需的。对于不同状态的染色质,核小体中会组装入相应的组蛋白变体,并且各种组蛋白变体的尾部也能发生多种修饰。这些变体通过改变核小体的空间构象和稳定性,决定基因转录的激活或沉默,DNA的修复,染色体的异染色化等。在组蛋白替换过程中,组蛋白变体是通过相应的染色质重构复合物组装入核小体,不同的变体有着不同的组装途径。对组蛋白变体的研究是近年来表观遗传学新的研究热点,也是对“组蛋白密码”的新的诠释。并且,组蛋白替换揭示了DNA-组蛋白相互作用变化的一种新的机制。

     

Histone proteomics and the epigenetic regulation of nucleosome mobility

Chromatin structure plays a vital role in the transmission of heritable gene expression patterns. The recent application of mass spectrometry to histone biology provides several striking insights into chromatin regulation. The continuing identification of new histone post-translational modifications is revolutionizing the ways in which we think about how access to genomic DNA is controlled. While post-translational modifications of the flexible histone tails continue to be an active area of investigation, the recent discovery of multiple modifications in the structured globular domains of histones provides new insights into how the nucleosome works. Recent experiments underscore the importance of a subgroup of these modifications: those that regulate histone-DNA interactions on the lateral surface of the nucleosome. This information highlights an emerging new paradigm in chromatin control, that of the epigenetic regulation of nucleosome mobility.     

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 H2A C-terminus regulates chromatin dynamics, remodeling, and histone H1 binding

The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.     

Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.     

DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal

Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed.