Nucleosome Core Research Articles

Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy.

DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose) polymerase 1) increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer) microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP)-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA.

Cryo-electron microscopy of chromatin biology.

The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM.

Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry.

Histone proteins are subject to dynamic post-translational modifications (PTMs) that cooperatively modulate the chromatin structure and function. Nearly all functional PTMs are found on the N-terminal histone domains (tails) of ∼50 residues protruding from the nucleosome core. Using high-definition differential ion mobility spectrometry (FAIMS) with electron transfer dissociation, we demonstrate rapid baseline gas-phase separation and identification of tails involving monomethylation, trimethylation, acetylation, or phosphorylation in biologically relevant positions. These are by far the largest variant peptides resolved by any method, some with PTM contributing just 0.25% to the mass. This opens the door to similar separations for intact proteins and in top-down proteomics.

Links between DNA methylation and nucleosome occupancy in the human genome

BackgroundDNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome.ResultsThe results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome.ConclusionsThus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

Functionalization of Ruthenium(II)(η6 -p-cymene)(3-hydroxy-2-pyridone) Complexes with (Thio)Morpholine: Synthesis and Bioanalytical Studies.

Hydroxypyr(id)ones constitute an emerging platform for the design of drug molecules, owing to their favorable biocompatibility and toxicity profiles. Herein, [RuII (η6 -p-cymene)] complexes with 3-hydroxy-2-pyridinone functionalized with morpholine and thiomorpholine, as a means often used in medicinal chemistry to alter the physicochemical properties of drug compounds, are reported. The compounds underwent hydrolysis of the Ru-Cl bond and the aqua species were stable for up to 48 h in aqueous solution, as observed by 1 H NMR spectroscopy and ESI-MS. The compounds formed adducts with amino acids and proteins through cleavage of the pyridinone ligand. Binding experiments to the nucleosome core particle by means of X-ray crystallography revealed similar reactivity and exclusive binding to histidine moieties of the histone proteins. Preliminary cyclin-dependent kinase 2 (CDK2)/cyclin A kinase inhibitory studies revealed promising activity similar to that of structurally related organometallic compounds.

Regulation Of Histone 3 Acetylation By Intermittent Hypoxia

Acetylation of nucleosome core histone proteins determines the structure of chromatin, which regulates the accessibility of DNA sequences to the transcriptional machinery. In general, histone hyperacetylation activates gene transcription and histone hypoacetylation leads to gene silencing. Intermittent hypoxia (IH), a hallmark feature of sleep apnea, induces transcriptional activation of several genes. Whether histone acetylation contributes to IH‐induced gene transcription is not known. The goal of the present study was to determine the effects of IH on histone acetylation and assess the underlying mechanisms. Experiments were performed on rat pheochromocytoma (PC12) cells exposed to alternating cycles of 1.5% O2 for 30 sec followed by 20% O2 for 5 min. Exposure of cells to 60 cycles of IH increased global acetylation of histone 3 (H3) but not histone 4 (H4) in a stimulus‐dependent and reversible manner compared to cell exposed to normoxia (20% O2). Specific lysine residues K9, K14, K18 and K27 contributed to increased H3 acetylation by IH. Histone deacetylase (HDAC) inhibitors mimicked IH‐increased H3 acetylation under normoxic conditions. IH reduced HDAC3 and HDAC5 protein expression. Pharmacological blockade of NADPH oxidase with triazolo pyrimidine (VAS‐2870), a specific Nox inhibitor inhibited the increased H3 acetylation and decreased HDAC expression that were induced by IH. These results suggest that ROS generated by NADPH oxidase triggers IH‐induced HDAC downregulation, leading to increased H3 acetylation.Support or Funding InformationSupported by NIH‐PO1‐HL90554.

Nucleosome Histone Tail Conformation and Dynamics: Impacts of Lysine Acetylation and a Nearby Minor Groove Benzo[a]pyrene-Derived Lesion.

Histone tails in nucleosomes play critical roles in regulation of many biological processes, including chromatin compaction, transcription, and DNA repair. Moreover, post-translational modifications, notably lysine acetylation, are crucial to these functions. While the tails have been intensively studied, how the structures and dynamics of tails are impacted by the presence of a nearby bulky DNA lesion is a frontier research area, and how these properties are impacted by tail lysine acetylation remains unexplored. To obtain molecular insight, we have utilized all atom 3 μs molecular dynamics simulations of nucleosome core particles (NCPs) to determine the impact of a nearby DNA lesion, 10S (+)-trans-anti-B[a]P-N2-dG-the major adduct derived from the procarcinogen benzo[a]pyrene-on H2B tail behavior in unacetylated and acetylated states. We similarly studied lesion-free NCPs to investigate the normal properties of the H2B tail in both states. In the lesion-free NCPs, charge neutralization upon lysine acetylation causes release of the tail from the DNA. When the lesion is present, it stably engulfs part of the nearby tail, impairing the interactions between DNA and tail. With the tail in an acetylated state, the lesion still interacts with part of it, although unstably. The lesion's partial entrapment of the tail should hinder the tail from interacting with other nucleosomes, and other proteins such as acetylases, deacetylases, and acetyl-lysine binding proteins, and thus disrupt critical tail-governed processes. Hence, the lesion would impede tail functions modulated by acetylation or deacetylation, causing aberrant chromatin structures and impaired biological transactions such as transcription and DNA repair.

Regulation of Nucleosome Stacking and Chromatin Compaction by the Histone H4 N-Terminal Tail–H2A Acidic Patch Interaction

Chromatin folding and dynamics are critically dependent on nucleosome–nucleosome interactions with important contributions from internucleosome binding of the histone H4 N-terminal tail K16-R23 domain to the surface of the H2A/H2B dimer. The H4 Lys16 plays a pivotal role in this regard. Using in vitro reconstituted 12-mer nucleosome arrays, we have investigated the mechanism of the H4 N-terminal tail in maintaining nucleosome–nucleosome stacking and mediating intra- and inter-array chromatin compaction, with emphasis on the role of K16 and the positive charge region, R17-R23. Analytical ultracentrifugation sedimentation velocity experiments and precipitation assays were employed to analyze effects on chromatin folding and self-association, respectively. Effects on chromatin folding caused by various mutations and modifications at position K16 in the H4 histone were studied. Additionally, using charge-quenching mutations, we characterized the importance of the interaction of the residues within the H4 positive charge region R17-R23 with the H2A acidic patch of the adjacent nucleosome. Furthermore, crosslinking experiments were conducted to establish the proximity of the basic tail region to the acidic patch. Our data indicate that the positive charge and length of the side chain of H4 K16 are important for its access to the adjacent nucleosome in the process of nucleosome–nucleosome stacking and array folding. The location and orientation of the H4 R17-R23 domain on the H2A/H2B dimer surface of the neighboring nucleosome core particle (NCP) in the compacted chromatin fiber were established. The dominance of electrostatic interactions in maintaining intra-array interaction was demonstrated.

Combined Monte Carlo/torsion-angle molecular dynamics for ensemble modeling of proteins, nucleic acids and carbohydrates

We describe a general method to use Monte Carlo simulation followed by torsion-angle molecular dynamics simulations to create ensembles of structures to model a wide variety of soft-matter biological systems. Our particular emphasis is focused on modeling low-resolution small-angle scattering and reflectivity structural data. We provide examples of this method applied to HIV-1 Gag protein and derived fragment proteins, TraI protein, linear B-DNA, a nucleosome core particle, and a glycosylated monoclonal antibody. This procedure will enable a large community of researchers to model low-resolution experimental data with greater accuracy by using robust physics based simulation and sampling methods which are a significant improvement over traditional methods used to interpret such data.

Evolutionary mechanism and biological functions of 8-mers containing CG dinucleotide in yeast.

The rules of k-mer non-random usage and the biological functions are worthy of special attention. Firstly, the article studied human 8-mer spectra and found that only the spectra of cytosine-guanine (CG) dinucleotide classification formed independent unimodal distributions when the 8-mers were classified into three subsets under 16 dinucleotide classifications. Secondly, the distribution rules were reproduced by other seven species including yeast, which showed that the evolution phenomenon had species universality. It followed that we proposed two theoretical conjectures: (1) CG1 motifs (8-mers including 1 CG) are the nucleosome-binding motifs. (2) CG2 motifs (8-mers including two or more than two CG) are the modular units of CpG islands. Our conjectures were confirmed in yeast by the following results: a maximum of average area under the receiver operating characteristic (AUC) resulted from CG1 information during nucleosome core sequences, and linker sequences were distinguished by three CG subsets; there was a one-to-one relationship between abundant CG1 signal regions and histone positions; the sequence changing of squeezed nucleosomes was relevant with the strength of CG1 signals; and the AUC value of 0.986 was based on CG2 information when CpG islands and non-CpG islands were distinguished by the three CG subsets.

Advances in phase plate cryo-EM imaging of DNA and nucleosomes

ABSTRACTContrast in electron cryo-microscopy (cryo-EM) is limited by the weak phase and radiation sensitive nature of biologic samples embedded in vitrified ice. We have recently shown that a new contrast enhancement technique utilizing the Volta phase plate can be combined with single particle analysis to determine the structure of a small chromatin complex, the nucleosome core particle, at near-atomic resolution. Here, we discuss advantages and limitations of the technique in terms of data collection, particle detection, and visualization of individual DNA molecules and higher-order chromatin structure.

Understanding Chromatin Structure and Dynamics: A Molecular Dynamics Study

Chromatin fibers are a complex of DNA and proteins, which are the building blocks of chromosomes within the nucleus of eukaryotic cells. They consist of repeating units of the nucleosome core particle (NCP), which consists of ∼147 DNA base pairs densely wrapped around a histone octamer. The structure and dynamics of poly-NCP arrays therefore directly influences several processes, including gene expression, DNA replication, and DNA repair. Here, we report results from a series of fully atomistic molecular dynamics simulations of polynucleosomal arrays. Analyses focuses on the stability and conformational dynamics of the chromatin fiber, along with the importance of stacking interactions between adjacent nucleosomes and inter-NCP contacts formed by the of histone tails. These results build on previous experimental and simulation results of single NCP systems, and shed new light on the relationship between the structure, function, and dynamics of chromatin fibers.

The Free-Energy Landscape of the di-Nucleosome: A Critical Role of the H4 Tails for the Nucleosome-Nucleosome Conformation

Studying nucleosome-nucleosome interaction is critical to understand chromatin dynamics. Here, we carried out molecular dynamics simulations of stacked di-nucleosome with different locations of H4 tails of the two nucleosomes. Using an adaptively biased potential method the nucleosome core particles (NCPs) were separated from each other. This simulation generated a variety of orientations of the separated NCPs depending on the location of the H4 tails. The free-energy landscapes were investigated for each of models which had different orientations of the NCP-NCP. The force of attraction between the NCPs mainly depended on the way how the H4 tails attached to the DNA of the other nucleosome and the orientation of the two nucleosomes. A simulation for the model (in which the direction of the separation was parallel) without the H4 tails showed that the interfacial interaction between the NCPs (without the H4 tails) was a several kcal/mol and the force became repulsive in the long range, which indicates that the presence of the H4 tails is crucial for the attractive force. Additional simulations for the models (with and without the H4 tails) at a high ion concentration showed that ions reduced the interfacial interaction by screening the salt-bridges formed at the interface between the NCPs, and contributed to the attractive force by a several kcal/mol. We concluded that the H4 tails are critical for forming the attractive tail bridge with the DNA of the other NCP and for diversifying the orientation of the NCP-NCP conformation, which would contribute to the polymorphism of the chromatin structure.

Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation.

Post-translational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitin modification is one such modification. We previously reported on the use of a thiirane linker to introduce a 1,2-aminothiol moiety at a cysteine residue for native chemical ligation with peptide thioesters, which permitted isopeptide mimetics to be produced. In this report, we describe the preparation of the ubiquitylated full length histone H3 at the 18 position and the construction of tetranucleosomes with recombinant histones H2A, H2B, H4, and DNA, which are slightly more stable than those that are prepared without ubiquitin modification. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Confident gene activity prediction based on single histone modification H2BK5ac in human cell lines

BackgroundThe histones in the core of nucleosomes may be subject to covalent post-transcriptional modifications. These modifications are thought to correlate with and possibly affect various genomic functions, including transcription. Each modification may alone or in combination with other modifications influence or be influenced by transcription. We aimed to identify correlations between single modifications or combinations of modifications at specific nucleosome sized gene regions with transcription activity based on global histone modification and transcription data of human CD4+ T cells and three other human cell lines. Transcription activity was defined in a binary fashion as either on or off. The analysis was done using the Classification and Regression Tree (CART) data mining protocol, and the Multifactorial Dimensionality Reduction (MDR) method was performed to confirm the CART results. These powerful methods have not previously been used for analysis of histone modification data.ResultsWe showed that analysis of the single histone modification H2BK5ac at only four gene regions correctly predicted transcription activity status of over 75% of genes in CD4+ T-cells. The H2BK5ac modification status also had high power for prediction of gene transcription activity in the three other cell lines studied. The informative gene regions with the H2BK5ac modification were all positioned proximal to transcription initiation sites. The CART and MDR methods were appropriate tools for the analysis performed. In the study, we also developed a non-arbitrary protocol for binary classification of genes as transcriptionally active or inactive.ConclusionsThe importance of H2BK5ac modification with regards to transcription control has not previously been emphasized. Analysis of this single modification at only four nucleosome sized gene regions, all of which are at or proximal to transcription initiation, has high power for prediction of gene transcription activity.

Differential Ability of Five DNA Glycosylases to Recognize and Repair Damage on Nucleosomal DNA.

Damage to genomic DNA leads to mutagenesis and disease. Repair of single base damage is initiated by DNA glycosylases, the first enzymes in the base excision repair pathway. Although eukaryotic packaging of chromosomal DNA in nucleosomes is known to decrease DNA glycosylase efficiency, the impact on individual glycosylases is unclear. Here, we present a model system in which we examine the repair of site-specific base damage in well-characterized nucleosome core particles by five different DNA glycosylases. We find that DNA glycosylase efficiency on nucleosome substrates depends not only on the geometric orientation of the damaged base but also on its identity, as well as on the size, structure, and mechanism of the glycosylase. We show via molecular modeling that inhibition of glycosylase activity is largely due to steric obstruction by the nucleosome core.

DNA polymerase β uses its lyase domain in a processive search for DNA damage.

DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol β has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol β scans DNA using a processive hopping mechanism and has a mean search footprint of ∼24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol β. Application of our processive search assay into nucleosome core particles revealed that Pol β is not processive in the context of a nucleosome, and its single-turnover activity is reduced ∼500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol β mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol β.

Interdomain Communication of the Chd1 Chromatin Remodeler across the DNA Gyres of the Nucleosome

Chromatin remodelers use a helicase-like ATPase motor to reposition and reorganize nucleosomes along genomic DNA. Yet, how the ATPase motor communicates with other remodeler domains in the context of the nucleosome has so far been elusive. Here, we report for the Chd1 remodeler a unique organization of domains on the nucleosome that reveals direct domain-domain communication. Site-specific cross-linking shows that the chromodomains and ATPase motor bind to adjacent SHL1 and SHL2 sites, respectively, on nucleosomal DNA and pack against the DNA-binding domain on DNA exiting the nucleosome. This domain arrangement spans the two DNA gyres of the nucleosome and bridges both ends of a wrapped, ∼90-bp nucleosomal loop of DNA, suggesting a means for nucleosome assembly. This architecture illustrates how Chd1 senses DNA outside the nucleosome core and provides a basis for nucleosome spacing and directional sliding away from transcription factor barriers.

The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes

Nucleosomes form the unit structure of the genome in eukaryotes, thereby constituting a fundamental tenet of chromatin biology. In canonical nucleosomes, DNA wraps around the histone octamer in a left-handed toroidal ramp. Here, in single-molecule magnetic tweezers studies of chaperone-assisted nucleosome assembly, we show that the handedness of the DNA wrapping around the nucleosome core is intrinsically ambidextrous, and depends on the pre-assembly supercoiling state of the DNA, i.e., it is not uniquely determined by the octameric histone core. Nucleosomes assembled onto negatively supercoiled DNA are found to exhibit a left-handed conformation, whereas assembly onto positively supercoiled DNA results in right-handed nucleosomes. This intrinsic flexibility to adopt both chiralities is observed both for canonical H3 nucleosomes, and for centromere-specific variant CENP-A nucleosomes. These data support recent advances suggesting an intrinsic adaptability of the nucleosome, and provide insights into how nucleosomes might rapidly re-assemble after cellular processes that generate positive supercoiling in vivo.

Asymmetric unwrapping of nucleosomal DNA propagates asymmetric opening and dissociation of the histone core

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.