Multiscale Modeling of Nucleosome Dynamics

DNA methylation and histone tail modifications are interrelated mechanisms involved in a wide range of biological processes, and disruption of this crosstalk is linked to diseases such as acute myeloid leukemia. In addition, DNA methyltransferase 3A (DNMT3A) activity is modulated by several regulatory proteins, including p53 and thymine DNA glycosylase (TDG). However, the relative role of histone tails and regulatory proteins in the simultaneous coordination of DNMT3A activity remains obscure. We observed that DNMT3A binds H3 tails and p53 or TDG at distinct allosteric sites to form DNMT3A–H3 tail-p53 or –TDG multiprotein complexes. Functional characterization of DNMT3A–H3 tail-p53 or –TDG complexes on human-derived synthetic histone H3 tails, mononucleosomes, or polynucleosomes shows p53 and TDG play dominant roles in the modulation of DNMT3A activity. Intriguingly, this dominance occurs even when DNMT3A is actively methylating nucleosome substrates. The activity of histone modifiers is influenced by their ability to sense modifications on histone tails within the same nucleosome or histone tails on neighboring nucleosomes. In contrast, we show here that DNMT3A acts on DNA within a single nucleosome, on nucleosomal DNA within adjacent nucleosomes, and DNA not associated with the DNMT3A–nucleosome complex. Our findings have direct bearing on how the histone code drives changes in DNA methylation and highlight the complex interplay between histone tails, epigenetic enzymes, and modulators of enzymatic activity.

Conformational Sampling of Unmodified and Acetylated H3 Histone Tails on a Nucleosome by All-Atom Model Molecular Dynamics Simulations

Nucleosome Dynamics Studied by Single-Pair FRET and Computer Simulations

Histone tails harbor a plethora of post-translational modifications that direct the function of chromatin regulators, which recognize them through effector domains. Effector domain/histone interactions have been broadly studied, but largely using peptide fragments of histone tails. Here, we extend these studies into the nucleosome context and find that the conformation adopted by the histone H3 tails is inhibitory to BPTF PHD finger binding. Using NMR spectroscopy and MD simulations, we show that the H3 tails interact robustly but dynamically with nucleosomal DNA, substantially reducing PHD finger association. Altering the electrostatics of the H3 tail via modification or mutation increases accessibility to the PHD finger, indicating that PTM crosstalk can regulate effector domain binding by altering nucleosome conformation. Together, our results demonstrate that the nucleosome context has a dramatic impact on signaling events at the histone tails, and highlights the importance of studying histone binding in the context of the nucleosome.

Ionizing radiation induces DNA damage both directly and indirectly through hydroxyl radicals (•OH) generated by water radiolysis. The indirect action involves hydrogen abstraction from deoxyribose, leading to DNA damage such as base release and strand breaks. The probability of such events depends on the accessibility of specific hydrogen atoms to OH radicals. Previous computational studies have examined naked DNA fragments; however, the effect of nucleosomal organization has not been addressed. Here, we performed molecular dynamics (MD) simulations using LAMMPS (Large-scale Atomic Molecular Massively Parallel Simulator) to evaluate the accessibility of •OH to deoxyribose hydrogens in both 12-bp naked B-type DNA and nucleosomal DNA containing histone proteins. The accessibility to the hydrogens increased, following the order H1’ ≈ H2’ ≈ H3’ < H4’ < H5’, consistent with previous studies. Accessibility was approximately proportional to the solvent accessible surface area (SASA), supporting SASA as a practical predictor of accessibility even in base-dependent differences were observed. In nucleosomal DNA, accessibility of hydrogens facing the histone core was significantly reduced compared to solvent-facing hydrogens, despite comparable SASA values. This reduction suggests that histones physically hinder OH radical penetration. These findings highlight the importance of considering protein–DNA complexes when modeling indirect radiation effects.

The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in "activating" histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion.

DNA methylation plays a vital role in epigenetic regulation in both plants and animals, and typically occurs at the 5-carbon position of the cytosine pyrimidine ring within the CpG dinucleotide steps. Cytosine methylation can alter DNA's geometry, mechanical and physico-chemical properties - thus influencing the molecular signaling events vital for transcription, replication and chromatin remodeling. Despite the profound effect cytosine methylation can have on DNA, the underlying atomistic mechanisms remain enigmatic. Many studies so far have produced controversial findings on how cytosine methylation dictates DNA flexibility and accessibility, nucleosome stability and dynamics. Here, we review the most recent experimental and computational studies that provide precise characterization of structure and function of cytosine methylation and its versatile roles in modulating DNA mechanics, nucleosome and chromatin structure, stability and dynamics. Moreover, the review briefly discusses the relationship between DNA methylation and nucleosome positioning, and the crosstalk between DNA methylation and histone tail modifications.

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-μs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 A deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA(Phe), pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.

We have applied all atoms discrete molecular dynamics (DMD) based on a quasi-physical potential to study the flexibility of an extended set of proteins for which atomistic MD simulations are available. The method uses pure physical potentials supplemented by information on secondary structure and despite its simplicity is able to reproduce with good accuracy the dynamics of proteins in solution. The method presents a clear improvement with respect to coarse-grained methods based on structure potentials and opens the possibility to explore dynamics of proteins out from the equilibrium and to trace conformational changes induced by interaction of proteins with both small and macromolecular ligands.

Lipid aggregation into fluid bilayers is an essential process for sustaining life. Simplified models of lipid structure, which allow for long time scales or large length scales not obtainable with all-atom simulations, have recently been developed and show promise for describing lipid dynamics in biological systems. Here, we describe two simplified models, a reduced-lipid model and a bola-lipid model for thermophilic bacterial membranes, developed for use with the rapid discrete molecular dynamics simulation method. In the reduced-lipid model, we represent the lipid chain by a series of three beads interacting through pairwise discrete potentials that model hydrophobic attractions between hydrocarbon tails in implicit solvent. Our phase diagram recapitulates those produced by continuous potential models with similar coarse-grained lipid representations. We also find that phase transition temperatures for our reduced-lipid model are dependent upon the flexibility of the lipid chain, giving an insight into archaebacterial membrane stability and prompting development of a bola-lipid model specific for archaebacteria lipids. With both the reduced-lipid and bola-lipid model, we find that the reduced flexibility inherent in archaebacteria lipids yields more stable bilayers as manifested by increased phase transition temperatures. The results of these studies provide a simulation methodology for lipid molecules in biological systems and show that discrete molecular dynamics is applicable to lipid aggregation and dynamics.

The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET and a single-molecule FRET cyclization assay. We found that a single base-pair C-C mismatch enhances DNA bendability and nucleosome mechanical stability for the 601-nucleosome positioning sequence. An increase in force required for DNA unwrapping from the histone core is observed for single base-pair C-C mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single-nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression. Mechanical stability of an intact nucleosome, that is mismatch-free, is also dependent on the species as we find that yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes.

Changes in the epigenome induced by the environment have been documented in diverse animal phyla, ranging from insects to rodents to humans.These include chromatin remodeling, histone tail modifi cations, and DNA methylation, and more recently the list has expanded to encompass noncoding RNA and microRNA gene regulation (Matzke and Birchler 2005).Thus, it is increasingly recognized that exposure to chemical, nutritional, behavioral, and physical factors alters gene expression and affects health and disease not only through mutation of but also through modifi cation of the epigenome.Moreover, such exposures have been directly linked with subsequent disease formation through deregulation of epigenetic mechanisms.Unlike genetic mutations, these epigenetic changes are potentially reversible, providing a unique avenue to improve human health.Consequently research in epigenetics has increased dramatically in the last few years (Figure 1).The term "epigenetics" was popularized in the early 1940s by developmental biologist Conrad Waddington (1940) to explain "the interactions of genes with their environment, which bring the phenotype into being."In the 1970s, Holliday and Pugh (1975) fi rst proposed covalent chemical DNA modifi cations, including methylation of cytosine-guanine (CpG) dinucleotides, as the molecular mechanism to explain Waddington's hypothesis.The revelations several decades later that X inactivation in mammals and genomic imprinting are regulated by complex and multifactorial mechanisms (Monk 1988;Willard et al. 1993) resulted in an updated definition, describing epigenetics as heritable changes in gene expression that occur without a change in DNA sequence, including the modifi cation of DNA methylation and chromatin remodeling (Wolfe and Matzke 1999).The genomics revolution inspired the investigation of genome-wide rather than local gene analyses, and the term "epigenomics" was

Epigenetic effects of nano-sized materials

Post-translational histone modifications abound and regulate multiple nuclear processes. Most modifications are targeted to the amino-terminal domains of histones. Here we report the identification and characterization of acetylation of lysine 56 within the core domain of histone H3. In the crystal structure of the nucleosome, lysine 56 contacts DNA. Phenotypic analysis suggests that lysine 56 is critical for histone function and that it modulates formamide resistance, ultraviolet radiation sensitivity, and sensitivity to hydroxyurea. We show that the acetylated form of histone H3 lysine 56 (H3-K56) is present during interphase, metaphase, and S phase. Finally, reverse genetic analysis indicates that none of the known histone acetyltransferases is solely responsible for H3-K56 acetylation in Saccharomyces cerevisiae.