Core Histone Octamer Research Articles

Histone H4 proteoforms and post-translational modifications in the Mus musculus brain with quantitative comparison of ages and brain regions.

Histone proteins are essential to the regulation of the eukaryotic genome. Histone post-translational modifications (PTMs) and single-molecule combinations of these modifications (proteoforms) allow for the regulation of many DNA-templated processes, most notably transcription. Histone H4 is a part of the core histone octamer, which packages DNA into nucleosomes. Top-down proteomics allows for the inquiry of the epigenetic landscape with proteoform-level specificity. Although these approaches are well-demonstrated ex vivo, our knowledge of in vivo histone proteoform biology remains sparse. Here, we demonstrate the first in vivo quantitative top-down analysis of histone H4 and analyze the forebrains and hindbrains of differently aged mice. This reveals novel differences between the mouse forebrain and hindbrain and region-specific changes during adolescence in histone H4 PTMs and proteoforms. At 25days of age (P25), histone H4 of the hindbrain is more acetylated than the forebrain. At 47days of age (P47), there are fewer significant differences in histone H4 PTMs and their combinations between regions. Histone H4 of the forebrain is more acetylated in P47 than in P25 forebrains. Hindbrains exhibit the opposite difference with histone H4 of the P25 hindbrain being more acetylated than that of P47 hindbrains. These differences are mainly driven by less abundant hyperacetylated proteoforms. Transcription of histone acetyltransferases such as p300, CBP, and HAT1 is known to be higher in cortical neurons, consistent with the observed acetylation levels. Lysine 20 methylation (K20me1, K20me2, and K20me3) is notably invariant with brain region and age difference.

Galaxy Dnpatterntools for Computational Analysis of Nucleosome Positioning Sequence Patterns.

Nucleosomes are basic units of DNA packing in eukaryotes. Their structure is well conserved from yeast to human and consists of the histone octamer core and 147 bp DNA wrapped around it. Nucleosomes are bound to a majority of the eukaryotic genomic DNA, including its regulatory regions. Hence, they also play a major role in gene regulation. For the latter, their precise positioning on DNA is essential. In the present paper, we describe Galaxy dnpatterntools—software package for nucleosome DNA sequence analysis and mapping. This software will be useful for computational biologists practitioners to conduct more profound studies of gene regulatory mechanisms.

Acylation of key sites in the histone octamer core destabilizes nucleosome arrays

The nucleosome is the fundamental structure of DNA organization in eukaryotic cells. Post-translational histone modifications in nucleosomes strongly influence transcription, presumably through regulation of access to DNA. Although many post-translational modifications have been studied as elements of the “histone code,” most of the implications and functions of these modifications are not well known, and we do not understand which, if any, modifications have intrinsic effects apart from “readers.” A key modification is lysine acylation.

The FAcilitates Chromatin Transcription (FACT) complex: Its roles in DNA repair and implications for cancer therapy

Genomic DNA in the nucleus is wrapped around nucleosomes, a repeating unit of chromatin. The nucleosome, consisting of octamer of core histones, is a barrier for several cellular processes that require access to the naked DNA. The FAcilitates Chromatin Transcription (FACT), a histone chaperone complex, is involved in nucleosome remodeling via eviction or assembly of histones during transcription, replication, and DNA repair. Increasing evidence suggests that FACT plays an important role in multiple DNA repair pathways including transcription-coupled nucleotide excision repair (TC-NER) of UV-induced damage, DNA single- and double-strand breaks (DSBs) repair, and base excision repair (BER) of oxidized or alkylated damaged bases. Further, studies have shown overexpression of FACT in multiple types of cancer and its association with drug resistance and patients’ poor prognosis. In this review, we discuss how FACT is accumulated at the damage site and what functions it performs. We describe the known mechanisms by which FACT facilitates repair of different types of DNA damage. Further, we highlight the recent advances in a class of FACT inhibitors, called curaxins, which show promise as a new adjuvant therapy to sensitize multiple types of cancer to chemotherapy and radiation.

Characterizing circulating nucleosomes in the plasma of dogs with lymphoma

BackgroundNucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay.A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU.ResultsDogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas.ConclusionsThe Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Characterizing circulating nucleosomes in the plasma of dogs with hemangiosarcoma

BackgroundNucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death, chromatin fragmentation and release of nucleosomes into the blood. The Nu.Q™ platform measures circulating nucleosomes in the blood of humans that result from disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using this ELISA-based assay.Samples from 77 dogs with a confirmed diagnosis of hemangiosarcoma and 134 healthy controls were utilized for this study. The HSA samples were recruited from the Texas A&M University Small Animal Clinic (TAMU-SAC) or purchased from biobanks. All control samples were recruited from the TAMU-SAC.ResultsDogs with hemangiosarcoma had a 6.6-fold increase in their median plasma nucleosome concentrations compared to controls (AUC 92.9 %). Elevated nucleosome concentrations were seen at all stages of disease and nucleosome concentrations increased with the stage of the disease.ConclusionsPlasma nucleosome concentrations are a reliable way to differentiate dogs with hemangiosarcoma from healthy dogs. Further testing is underway to better characterize cancer associated HSA circulating nucleosomes and optimize future diagnostics for canine HSA detection.

Chromatin dynamics: Nucleosome occupancy and sensitivity as determinants of gene expression and cell fate.

The nucleosome, consisting of ~150bp of DNA wrapped around a core histone octamer, is a regulator of nuclear events that contributes to gene expression and cell fate. Nucleosome organization at promoters and their associated remodeling events are important regulators of access to the genome. Occupancy alone, however, is not the only nucleosomal characteristic that plays a role in genome regulation. Nucleosomes at the transcription start sites (TSSs) of genes show differential sensitivity to micrococcal nuclease (MNase) and this differential sensitivity is linked to transcription and regulatory factor binding events. Recently, lymphoblastoid cells treated with heat-killed Salmonella typhimurium were shown to exhibit increased MNase sensitivity specifically at genes implicated in immune responses. Increased sensitivity at the −1-nucleosome permitted transcription factor and RNA Pol II binding events. This system illustrates how cytoplasmic signals induce altered chromatin states to produce a specific cellular response to a stimulus. Innate immune activation is a longstanding model for inducible promoters, transcriptional activation, and differential nucleosomal sensitivity in response to immune activation and offers a model that may be largely applicable to other specific cellular responses including viral infection and cancer. Previous work has shown that early transformation events are associated with prolonged nucleosome occupancy changes that are not observed later in cancer progression. Herein, we propose a model in which we suggest that detailed studies of nucleosomal occupancy and sensitivity in response to specific stimuli will provide insight into the regulation of nuclear events in cancer and other biological processes.

Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer.

Nucleosomes consist of small fragments of DNA wrapped around a histone octamer core. Diseases such as cancer or inflammation lead to cell death, which causes fragmentation and release of nucleosomes into the blood. The Nu.Q™ technology measures circulating nucleosome levels and exploits the different compositions of cancer derived nucleosomes in blood to detect and identify cancer even at early stages. The objectives of this study are to identify the optimal sample type for the Nu.Q™ H3.1 assay and to determine if it can accurately detect nucleosomes in the blood of healthy canines as well as those with cancer. Blood samples from healthy canine volunteers as well as dogs newly diagnosed with lymphoma were used. The blood was processed at a variety of times under a variety of conditions to determine the most reliable sample type and conditions, and to develop an appropriate processing strategy to ensure reliably accurate results. Nucleosomes could be detected using a variety of sample collection and processing protocols. Nucleosome signals were highest in EDTA plasma and serum samples and most consistent in plasma. Samples should be processed within an hour of collection. Experiments showed that samples were able to withstand several freeze thaw cycles. Processing time and tcollection tube type did affect nucleosome detection levels. Finally, significantly elevated concentrations of nucleosomes were seen in a small cohort of dogs that had been newly diagnosed with lymphoma. When samples are collected and processed appropriately, the Nu.Q™ platform can reliably detect nucleosomes in the plasma of dogs. Further testing is underway to validate and optimize the Nu.Q™ platform for veterinary use.

P12. Abstract Title: The Effects of Glycosaminoglycans on DNasel-Mediated Degradation of Neutrophil Extracellular Traps and DNA-Histone Complexes

<h3>Background</h3> Neutrophil extracellular traps (NETs), released from activated neutrophils, have been implicated in contributing to micro- and macro-vascular thrombosis. The major structural component of NETs is nucleosomes, which consists of DNA wrapped around a core histone octamer containing two copies each of histone H2A, H2B, H3, and H4. The release of NETs occurs via a unique cell death pathway (termed NETosis), which requires the decondensation of chromatin by PAD4. PAD4 is a nuclear enzyme that converts specific arginine residues to citrulline on histones, thereby reducing the positive charge of histones. DNA activates the intrinsic pathway of coagulation and impairs fibrinolysis, whereas histones activate platelets and are cytotoxic to vascular endothelial cells. In purified systems, DNA-histone complexes are resistant to digestion by DNaseI. The addition of unfractionated heparin (UFH) displaces the DNA-histone complex, thereby rendering DNA susceptible to digestion by DNaseI. <h3>Objectives</h3> The objective of this study is to examine the effects of various glycosaminoglycans (GAGs), including UFH (15kDa), Enoxaparin (4.5kDa), and Fondaparinux (1.7kDa) on DNaseI-mediated digestion of DNA-protein complexes as well as NETs released by activated neutrophils. <h3>Methods</h3> Genomic DNA was isolated from human leukocytes and incubated with a mixture of bovine histones. The DNA protein complexes were treated with recombinant human DNaseI (Pulmozyme) in the absence or presence of GAGs and visualized using agarose gel electrophoresis. To produce NETs, neutrophils from healthy volunteers were added to glass coverslips and stimulated with PMA for 4 hours. The NETs were then treated with DNaseI in the absence or presence of GAGs and visualized using fluorescence microscopy. The binding affinities between the different GAGs and histones were determined by Bio-layer Interferometry (BLItz instrument). <h3>Results</h3> DNaseI alone was unable to degrade DNA-histone complexes. The addition of UFH or Enoxaparin rendered the complexes susceptible to complete digestion by DNaseI, whereas Fondaparinux did not. Next, we measured the binding affinities between the GAGs and individual histones. We found that UFH and Enoxaparin bind histones H2A, H2B, H3, and H4 with high affinities (KD values ranging from 1 pM to 6.2 nM), which supports the model that these GAGs can displace histones from DNA-histone complexes. With respect to NETs released by activated neutrophils, DNaseI alone was able to completely digest the DNA component of NETs even in the absence of GAGs. <h3>Conclusions</h3> Our studies suggest that GAGs displace DNA-histone complexes, thereby rendering DNA susceptible to degradation by DNaseI. The inability of Fondaparinux to displace the DNA-histone complexes suggests that the GAG cofactor activity is size-dependent. In contrast, DNaseI is able to degrade DNA within NETs without a requirement for GAGs. The ability of DNaseI to degrade DNA within NETs may be due to histone citrullination that occurs during NETosis, a process that weakens DNA-histone interactions.

Chromatin Folding Heterogeneity Inferred from High-Resolution Nucleosome Structures

DNA in eukaryotic cell nuclei occurs in a highly compact chromatin structure where the basic packaging unit is the nucleosome. This dynamic structure displays a wide range in compaction and provides a regulatory mechanism for many biologically relevant processes, such as gene expression. Since the first description of nucleosome-decorated DNA as beads on a string condensing into a 30 nm fiber in vitro, there have been many attempts to understand the nature of chromatin compaction and to create models at this scale. However, the proposed models for the 3D organization of the nucleosomes and intervening DNA vary quite significantly, and the very existence and relevance of a 30 nm structure in vivo has been highly debated. An analysis of the available high-resolution nucleosome core particle (NCP) structures shows subtle, yet significant differences in DNA wrapping around the histone octamer core, potentially due to DNA and histone modifications, presence of NCP-binding proteins, experimental crystallization/cryoEM conditions, intrinsic flexibility, etc. Monte Carlo simulations of regular nucleosomal arrays using a meso-scale representation of chromatin suggest that these local differences can result in large changes in global structure, comparable to the effect of changes in nucleosome spacing by about 2-3 base pairs. Our results suggest that a regular nucleosome array with a 177-base pair repeat can display either a loose three-stack or a more compact two-stack arrangement, depending on the mode of DNA wrapping in the nucleosome. Insights from our work may help bridge the significant divide in the proposed chromatin models based on various experimental results.

Distortion of histone octamer core promotes nucleosome mobilization by a chromatin remodeler.

Adenosine 5'-triphosphate (ATP)-dependent chromatin remodeling enzymes play essential biological roles by mobilizing nucleosomal DNA. Yet, how DNA is mobilized despite the steric constraints placed by the histone octamer remains unknown. Using methyl transverse relaxation-optimized nuclear magnetic resonance spectroscopy on a 450-kilodalton complex, we show that the chromatin remodeler, SNF2h, distorts the histone octamer. Binding of SNF2h in an activated ATP state changes the dynamics of buried histone residues. Preventing octamer distortion by site-specific disulfide linkages inhibits nucleosome sliding by SNF2h while promoting octamer eviction by the SWI-SNF complex, RSC. Our findings indicate that the histone core of a nucleosome is more plastic than previously imagined and that octamer deformation plays different roles based on the type of chromatin remodeler. Octamer plasticity may contribute to chromatin regulation beyond ATP-dependent remodeling.

Nucleosome Dynamics at Microsecond Timescale: DNA-Protein Interactions, Water-Mediated Interactions and Nucleosome Formation

At the cornerstone of eukaryotic chromatin is the association of DNA with histone proteins to form nucleosomes. Every nucleosome core particle consists of 147 bp of DNA wrapped around and octamer of histones. Many factors (such as DNA sequence, histone variants, post-translational modifications, etc.) can affect the stability and dynamics of nucleosomes, which in turn provide key mechanisms for epigenetic regulation of gene expression. We report an extensive study of nucleosome dynamics and organization via molecular modeling based on all-atom microsecond molecular dynamics simulations. We analyze dynamics of full nucleosomes including linker DNA segments and full-length histones in explicit solvent and compare it to the dynamics of nucleosome core particle and histone octamer alone. Extensively long simulation time scale allows us to address the questions of conformation coupling between histones and DNA as well as study the interaction patterns of flexible histone tails. Detailed analysis of protein-DNA interactions is performed including water-mediated interactions and the role of water molecules in the central nucleosome pore. We analyze the rearrangement of nucleosome structure upon DNA binding and discuss the cooperativity of interactions in nucleosome formation. This work was supported by the Intramural Research Programs of NLM and NCI; and RSF grant No. 14-24-00031 (nucleosome visualization algorithms).

A New Histone Structure Which Binds DNA at Its Eight Subunit N-Termini

A new model for the nucleosome is presented. The histone octamer core is unchanged, but the location of the DNA is different. Since the highest number, and highest concentration of positively- charged amino acid residues is located not in the “superhelical...

AFM studies in diverse ionic environments of nucleosomes reconstituted on the 601 positioning sequence.

Atomic force microscopy (AFM) was used to study mononucleosomes reconstituted from a DNA duplex of 353bp containing the strong 601 octamer positioning sequence, together with recombinant human core histone octamers. Three parameters were measured: 1) the length of DNA wrapped around the core histones; 2) the number of superhelical turns, calculated from the total angle through which the DNA is bent, and 3) the volume of the DNA-histone core. This approach allowed us to define in detail the structural diversity of nucleosomes caused by disassembly of the octasome to form subnucleosomal structures containing hexasomes, tetrasomes and disomes. At low ionic strength (TE buffer) and in the presence of physiological concentrations of monovalent cations, the majority of the particles were subnucleosomal, but physiological concentrations of bivalent cations resulted in about half of the nucleosomes being canonical octasomes in which the exiting DNA duplexes cross orthogonally. The dominance of this last species explains why bivalent but not monovalent cations can induce the initial step towards compaction and convergence of neighboring nucleosomes in nucleosomal arrays to form the chromatin fiber in the absence of linker histone. The observed nucleosome structural diversity may reflect the functional plasticity of nucleosomes under physiological conditions.

Novel nucleosomal particles containing core histones and linker DNA but no histone H1.

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.

Chromatin—a global buffer for eukaryotic gene control

Most of eukaryotic DNA is embedded into nucleosome arrays formed by DNA wrapped around a core histone octamer. Nucleosome is a fundamental repeating unit of chromatin guarding access to the genetic information. Here, I will discuss two facets of nucleosome in eukaryotic gene control. On the one hand, nucleosome acts as a regulatory unit, which controls gene switches through a set of post-translational modifications occurring on histone tails. On the other hand, global configuration of nucleosome arrays with respect to nucleosome positioning, spacing and turnover acts as a tuning parameter for all genomic functions. A “histone code” hypothesis extents the Jacob-Monod model for eukaryotic gene control; however, when considering factors capable of reconfiguring entire nucleosome array, such as ATP-dependent chromatin remodelers, this model becomes limited. Global changes in nucleosome arrays will be sensed by every gene, yet the transcriptional responses might be specific and appear as gene targeted events. What determines such specificity is unclear, but it’s likely to depend on initial gene settings, such as availability of transcription factors, and on configuration of new nucleosome array state.

Changes in nucleosome occupancy occur in a chromosome specific manner.

In the eukaryotic nucleus, DNA is packaged into chromatin. The fundamental subunit of chromatin is the nucleosome, DNA is wrapped 1.6 times around a histone octamer core. Nuclear processes in eukaryotes are impacted by whether regulatory DNA is occupied by nucleosomes. We used microarrays to measure nucleosome occupancy in human cells post-Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation at hundreds of immunity-related loci. The detailed analysis of these technologies can be found in recent publications from our lab [1,3]. We found that nucleosome redistributions displayed chromosome specific nucleosome occupancy. This resource can be used to map nucleosome distributions in a variety of biological contexts.

Structure of the JmjC-domain-containing protein JMJD5

The post-translational modification of histone tails is the principal process controlling epigenetic regulation in eukaryotes. The lysine methylation of histones is dynamically regulated by two distinct classes of enzymes: methyltransferases and demethylases. JMJD5, which plays an important role in cell-cycle progression, circadian rhythms and embryonic cell proliferation, has been shown to be a JmjC-domain-containing histone demethylase with enzymatic activity towards H3K36me2. Here, the crystal structure of human JMJD5 lacking the N-terminal 175 amino-acid residues is reported. The structure showed that the Gln275, Trp310 and Trp414 side chains might block the insertion of methylated lysine into the active centre of JMJD5, suppressing the histone demethylase activity of the truncated JMJD5 construct. A comparison of the structure of JMJD5 with that of FIH, a well characterized protein hydroxylase, revealed that human JMJD5 might function as a protein hydroxylase. The interaction between JMJD5 and the core histone octamer proteins indicated that the histone proteins could be potential substrates for JMJD5.

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.