Chromatin Fibers Research Articles

Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients

Background Neutrophil extracellular traps (NETs) are decondensed chromatin fibers that might play a role in the prothrombotic state of cancer patients. Objectives To investigate whether the levels of citrullinated histone H3 (H3Cit), a biomarker for NET formation, cell-free DNA (cfDNA) and nucleosomes predict venous thromboembolism (VTE) in cancer patients. Patients/Methods Nine-hundred and forty-six patients with newly diagnosed cancer or progression after remission were enrolled in this prospective observational cohort study. H3Cit, cfDNA and nucleosome levels were determined at study inclusion, and patients were followed for 2 years. VTE occurred in 89 patients; the cumulative 3-month, 6-month, 12-month and 24-month incidence rates of VTE were 3.7%, 6.0%, 8.1%, and 10.0%, respectively. Results Patients with elevated H3Cit levels (> 75th percentile of its distribution, n = 236) experienced a higher cumulative incidence of VTE (2-year risk of 14.5%) than patients with levels below this cut-off (2-year risk of 8.5%, n = 710). In a competing-risk regression analysis, a 100 ng mL-1 increase in H3Cit level was associated with a 13% relative increase in VTE risk (subdistribution hazard ratio [SHR] 1.13, 95% confidence interval [CI] 1.04-1.22). This association remained after adjustment for high VTE risk and very high VTE risk tumor sites, D-dimer level, and soluble P-selectin level (SHR 1.13, 95% CI 1.04-1.22). The association of elevated nucleosome and cfDNA levels with VTE risk was time-dependent, with associations with a higher risk of VTE only during the first 3-6 months. Conclusion These data suggest that biomarkers of NET formation are associated with the occurrence of VTE in cancer patients, indicating a role of NETs in the pathogenesis of cancer-associated thrombosis.

TADs are 3D structural units of higher-order chromosome organization in Drosophila.

Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.

Impact of the Linker Histone H1 on the Structure and Dynamics of Chromatin Fibers: A Molecular Dynamics Study

Impact of the Linker Histone H1 on the Structure and Dynamics of Chromatin Fibers: A Molecular Dynamics Study

Linker DNA Length Defines the Structure of Chromatin Fibers

In eukaryotes, DNA is compacted into chromatin and the mechanical and structural properties of chromatin fibers play an important role in gene regulation. For well-defined, in vitro reconstituted nucleosomal arrays it has been shown that their higher order structure depends on the length of the linker DNA. Most studies however, focused on linker length consisting of multiples of 10 base pairs and report zig-zag or a solenoidal structures of stacked nucleosomes. In nature however, linker DNA lengths of 10n+5 base pairs are most abundant. For short linkers fibers an alternative anti-parallel zig-zag structure was recently proposed, but experimental evidence is lacking. Here, we used multiplexed magnetic tweezers to measure mechanical parameters of fibers with 20, 25, 50 and 55 bp linker DNA. Based on the stiffness and the stacking energy, we conclude that the 10n+5 fibers fold differently than 10n fibers, indicating a dominant role of the linker DNA on chromatin folding and consequently epigenetic regulation. These results suggest that nucleosome phasing should be controlled with base pair accuracy in order to create specific higher order chromatin structures.

Single Molecule Observation of Chromatin Compaction Reveals the Role of Histone Tails and Their Epigenetic Modifications

The basic unit of DNA compaction in eukaryotic cells is the nucleosome, which is composed of DNA wrapped around histone octamer (H2A, H2B, H3, H4), and the control of its wrapping-unwrapping, the repositioning of the histone octamer, and the compaction of the nucleosome array is central to the gene regulation mechanism. However, the mechanistic aspect of such regulation mechanism is yet to be clearly understood. The nucleosomes dynamically form higher order structure of chromatin fibers to achieve more compact state. Cells control the structure of chromatin fibers by introducing isoforms of histone, modifications on histone tails, or change in nucleosome spacing, in order to regulate the accessibility of the DNA to various nuclear proteins. Using magnetic tweezers combined with simultaneous single molecule fluorescence detection, we measured the compaction-decompaction dynamics of nucleosome arrays. By controlling the DNA sequence composition and spacer length, making tail-deleted histone mutants, and also introducing site-specific epigenetic modification to the tail of histone H3, we revealed their interplays to control the compaction of chromatin and histone unwrapping. Here we present our preliminary results on these attempts.

Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1\u03b1

The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

Contribution of transposable elements and distal enhancers to evolution of human-specific features of interphase chromatin architecture in embryonic stem cells.

Transposable elements have made major evolutionary impacts on creation of primate-specific and human-specific genomic regulatory loci and species-specific genomic regulatory networks (GRNs). Molecular and genetic definitions of human-specific changes to GRNs contributing to development of unique to human phenotypes remain a highly significant challenge. Genome-wide proximity placement analysis of diverse families of human-specific genomic regulatory loci (HSGRL) identified topologically associating domains (TADs) that are significantly enriched for HSGRL and designated rapidly evolving in human TADs. Here, the analysis of HSGRL, hESC-enriched enhancers, super-enhancers (SEs), and specific sub-TAD structures termed super-enhancer domains (SEDs) has been performed. In the hESC genome, 331 of 504 (66%) of SED-harboring TADs contain HSGRL and 68% of SEDs co-localize with HSGRL, suggesting that emergence of HSGRL may have rewired SED-associated GRNs within specific TADs by inserting novel and/or erasing existing non-coding regulatory sequences. Consequently, markedly distinct features of the principal regulatory structures of interphase chromatin evolved in the hESC genome compared to mouse: the SED quantity is 3-fold higher and the median SED size is significantly larger. Concomitantly, the overall TAD quantity is increased by 42% while the median TAD size is significantly decreased (p= 9.11E-37) in the hESC genome. Present analyses illustrate a putative global role for transposable elements and HSGRL in shaping the human-specific features of the interphase chromatin organization and functions, which are facilitated by accelerated creation of novel transcription factor binding sites and new enhancers driven by targeted placement of HSGRL at defined genomic coordinates. A trend toward the convergence of TAD and SED architectures of interphase chromatin in the hESC genome may reflect changes of 3D-folding patterns of linear chromatin fibers designed to enhance both regulatory complexity and functional precision of GRNs by creating predominantly a single gene (or a set of functionally linked genes) per regulatory domain structures. Collectively, present analyses reveal critical evolutionary contributions of transposable elements and distal enhancers to creation of thousands primate- and human-specific elements of a chromatin folding code, which defines the 3D context of interphase chromatin both restricting and facilitating biological functions of GRNs.

Estimation of the Spatial Chromatin Structure Based on a Multiresolution Bead-Chain Model

We present a method to infer 3D chromatin configurations from Chromosome Conformation Capture data. Quite a few methods have been proposed to estimate the structure of the nuclear dna in homogeneous populations of cells from this kind of data. Many of them transform contact frequencies into euclidean distances between pairs of chromatin fragments, and then reconstruct the structure by solving a distance-to-geometry problem. To avoid inconsistencies, our method is based on a score function that does not require any frequency-to-distance translation. We propose a multiscale chromatin model where the chromatin fiber is suitably partitioned at each scale. The partial structures are estimated independently, and connected to rebuild the whole fiber. Our score function consists of a data-fit part and a penalty part, balanced automatically at each scale and each subchain. The penalty part enforces soft geometric constraints. As many different structures can fit the data, our sampling strategy produces a set of solutions with similar scores. The procedure contains a few parameters, independent of both the scale and the genomic segment treated. The partition of the fiber, along with intrinsically parallel parts, make this method computationally efficient. Results from human genome data support the biological plausibility of our solutions.

Probing Chromatin Structure with Magnetic Tweezers.

Magnetic tweezers form a unique tool to study the topology and mechanical properties of chromatin fibers. Chromatin is a complex of DNA and proteins that folds the DNA in such a way that meter-long stretches of DNA fit into the micron-sized cell nucleus. Moreover, it regulates accessibility of the genome to the cellular replication, transcription, and repair machinery. However, the structure and mechanisms that govern chromatin folding remain poorly understood, despite recent spectacular improvements in high-resolution imaging techniques. Single-molecule force spectroscopy techniques can directly measure both the extension of individual chromatin fragments with nanometer accuracy and the forces involved in the (un)folding of single chromatin fibers. Here, we report detailed methods that allow one to successfully prepare in vitro reconstituted chromatin fibers for use in magnetic tweezers-based force spectroscopy. The higher-order structure of different chromatin fibers can be inferred from fitting a statistical mechanics model to the force-extension data. These methods for quantifying chromatin folding can be extended to study many other processes involving chromatin, such as the epigenetic regulation of transcription.

Asymmetric Histone Incorporation During DNA Replication in <i>Drosophila </i>Male Germline Stem Cells

Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell. We previously reported that in male Drosophila germline stem cells (GSCs), preexisting (old) histone H3 is segregated to the self-renewed stem cell, whereas the daughter incorporates newly synthesized (new) H3. Here, we show that histone H4 is likewise segregated asymmetrically, while histones H2A and H2B are segregated symmetrically. Using superresolution imaging, we visualize spatially separable old and new H3 distributions in interphase GSC nuclei and on isolated replicating chromatin fibers. Furthermore, using both superresolved chromatin fibers and proximity ligation assay, we demonstrate that old H3 are preferentially retained on the leading strand while new H3 preferentially associate with the lagging strand. Finally, using a dual nucleoside analog incorporation assay, the temporal separation between leading strand and lagging strand synthesis is detectable. Together, these results demonstrate that the spatial and temporal asymmetries inherent to DNA replication may serve to bias histone incorporation, suggesting an unappreciated role for DNA replication in asymmetrically dividing cells.

Ultrastructure Based Morphofunctional Variation of Olfactory Crypt Neuron in a Monomorphic Protogynous Hermaphrodite Mudskipper (Gobiidae: Oxudercinae) (Pseudapocryptes lanceolatus [Bloch and Schneider]).

Pseudapocryptes lanceolatus (Bloch and Schneider) is a monomorphic protogynous hermaphrodite teleost that possesses ovotestis as gonadal unit of reproductive structure. At the onset of breeding season (i.e., June–July), the ovarian tissue is gradually differentiating into female-phased P. lanceolatus. At the same time, the pear-shaped crypt cells (a type of neuron) are frequently appeared at apical part of pseudostratified olfactory neuroepithelium of P. lanceolatus. The crypt neuron is characterized by the presence of sunken cilia and microvilli at the proximal region. The features of subcellular organelles are also explored in lieu of their probable functional significance. The nucleoplasm of mature crypt neuron shows chromatin granules having diameter: 15–25 nm. This cell undergoes neural apoptosis at the end of breeding phase (i.e., October–November). Fragmented chromatin fibers with numerous chromatin granules (diameter: 25–30 nm) in nucleoplasm and lysosomal diversity are the most notable characters of apoptotic crypt neuron. The large accumulation of heterochromatin chromatins in nucleoplasm is also marked under fluorescence microscope. The frequent presence of acetylcholinesterase-positive vesicles in axoplasm of crypt neurons is also a prime subcellular indicator for inhibition of neural transmission of olfactory signals. Therefore, it is concluded that the sex differentiation in P. lanceolatus and occurrence of crypt neuron in olfactory neuroepithelium are interrelated events during the reproductive period. Consequently, we hypothesized that the crypt neuron plays an active role in the implementation of unique reproductive strategy through recognition of pheromonal cues within the social organization of P. lanceolatus.

A Method for Extracting the Nuclear Scaffold from the Chromatin Network.

Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the observation that, upon its extraction from the nuclei, the remaining nuclear chromatins were liberated into extended and often parallel chromatin fibers. Basically, this protocol includes the generation of pure nuclei, treatment of the nuclei with Triton X-100 to generate envelope-depleted nuclei (TxN), and extraction of the nuclei at 500 mM NaCl in a sucrose-containing buffer. This combined extract of TxN is known as TxNE.

Timoshenko beam model for chiral materials

Natural and artificial chiral materials such as deoxyribonucleic acid (DNA), chromatin fibers, flagellar filaments, chiral nanotubes, and chiral lattice materials widely exist. Due to the chirality of intricately helical or twisted microstructures, such materials hold great promise for use in diverse applications in smart sensors and actuators, force probes in biomedical engineering, structural elements for absorption of microwaves and elastic waves, etc. In this paper, a Timoshenko beam model for chiral materials is developed based on noncentrosymmetric micropolar elasticity theory. The governing equations and boundary conditions for a chiral beam problem are derived using the variational method and Hamilton’s principle. The static bending and free vibration problem of a chiral beam are investigated using the proposed model. It is found that chirality can significantly affect the mechanical behavior of beams, making materials more flexible compared with nonchiral counterparts, inducing coupled twisting deformation, relatively larger deflection, and lower natural frequency. This study is helpful not only for understanding the mechanical behavior of chiral materials such as DNA and chromatin fibers and characterizing their mechanical properties, but also for the design of hierarchically structured chiral materials.

The 10-nm chromatin fiber and its relationship to interphase chromosome organization.

A chromosome is a single long DNA molecule assembled along its length with nucleosomes and proteins. During interphase, a mammalian chromosome exists as a highly organized supramolecular globule in the nucleus. Here, we discuss new insights into how genomic DNA is packaged and organized within interphase chromosomes. Our emphasis is on the structural principles that underlie chromosome organization, with a particular focus on the intrinsic contributions of the 10-nm chromatin fiber, but not the regular 30-nm fiber. We hypothesize that the hierarchical globular organization of an interphase chromosome is fundamentally established by the self-interacting properties of a 10-nm zig-zag array of nucleosomes, while histone post-translational modifications, histone variants, and chromatin-associated proteins serve to mold generic chromatin domains into specific structural and functional entities.

Nucleosomes Exhibit Non-uniform Unwrapping Along Native Chromatin Fibers with Increasing Salt Concentration as Revealed by Direct Imaging in a Microfluidic Channel.

Identifying the distribution of the higher-order structure of chromatin - a complex of DNA and proteins - along genomic DNA can clarify the mechanisms underlying cell development and differentiation, including gene regulation. However, genome-wide analysis of this distribution at the single-cell level remains an outstanding challenge. Here, the authors report a new method for investigating changes in and the distribution of higher-order structures along native chromatin fibers - ranging over 100 µm in length - relative to changes in salt concentration. To this end, the authors developed a microfluidic platform that enabled us to isolate chromatin fibers from single cells and tether them to microstructures in a microfluidic channel without fragmentation. The fibers were then exposed to varying concentrations of salt solution under microscopic observation. As a result, the fibers are non-uniformly elongated by up to 2-3 times along the fiber axis as salt concentration was increased from 0 to 3 M, suggesting that chromosome structural stability is non-uniformly distributed along chromatin fibers in their native form. Thus, our system enables direct microscopic analysis of individual chromatin fibers from single cells, which can provide insights into epigenetic mechanisms of cell development, cell differentiation, and carcinogenesis.

Nucleosome acidic patch-targeting binuclear ruthenium compounds induce aberrant chromatin condensation

The ‘acidic patch’ is a highly electronegative cleft on the histone H2A–H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications in drug development and as tools for chromatin research.

DNA knots occur in intracellular chromatin.

In vivo DNA molecules are narrowly folded within chromatin fibers and self-interacting chromatin domains. Therefore, intra-molecular DNA entanglements (knots) might occur via DNA strand passage activity of topoisomerase II. Here, we assessed the presence of such DNA knots in a variety of yeast circular minichromosomes. We found that small steady state fractions of DNA knots are common in intracellular chromatin. These knots occur irrespective of DNA replication and cell proliferation, though their abundance is reduced during DNA transcription. We found also that in vivo DNA knotting probability does not scale proportionately with chromatin length: it reaches a value of ∼0.025 in domains of ∼20 nucleosomes but tends to level off in longer chromatin fibers. These figures suggest that, while high flexibility of nucleosomal fibers and clustering of nearby nucleosomes facilitate DNA knotting locally, some mechanism minimizes the scaling of DNA knot formation throughout intracellular chromatin. We postulate that regulation of topoisomerase II activity and the fractal architecture of chromatin might be crucial to prevent a potentially massive and harmful self-entanglement of DNA molecules in vivo.

Single-molecule compaction of megabase-long chromatin molecules by multivalent cations.

To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250–400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo. Variation of HO loading revealed a number of unique features related to the efficiency of chromatin compaction by multivalent cations, the mechanism of compaction, and the character of partly compact chromatin structures. The observations may be relevant for how DNA accessibility in chromatin is maintained. Compaction of saturated chromatin, in turn, is accompanied by an intra-chain segregation at the level of single chromatin molecules, suggesting an intriguing scenario of selective activation/deactivation of DNA as a result of chromatin fiber heterogeneity due to the nucleosome positioning. We suggest that this chromatin, reconstituted on megabase-long DNA because of its large size, is a useful model of eukaryotic chromatin.

Transcription-induced supercoiling as the driving force of chromatin loop extrusion during formation of TADs in interphase chromosomes.

Using molecular dynamics simulations, we show here that growing plectonemes resulting from transcription-induced supercoiling have the ability to actively push cohesin rings along chromatin fibres. The pushing direction is such that within each topologically associating domain (TAD) cohesin rings forming handcuffs move from the source of supercoiling, constituted by RNA polymerase with associated DNA topoisomerase TOP1, towards borders of TADs, where supercoiling is released by topoisomerase TOPIIB. Cohesin handcuffs are pushed by continuous flux of supercoiling that is generated by transcription and is then progressively released by action of TOPIIB located at TADs borders. Our model explains what can be the driving force of chromatin loop extrusion and how it can be ensured that loops grow quickly and in a good direction. In addition, the supercoiling-driven loop extrusion mechanism is consistent with earlier explanations proposing why TADs flanked by convergent CTCF binding sites form more stable chromatin loops than TADs flanked by divergent CTCF binding sites. We discuss the role of supercoiling in stimulating enhancer promoter contacts and propose that transcription of eRNA sends the first wave of supercoiling that can activate mRNA transcription in a given TAD.

Epigenetic Transitions and Knotted Solitons in Stretched Chromatin

The spreading and regulation of epigenetic marks on chromosomes is crucial to establish and maintain cellular identity. Nonetheless, the dynamic mechanism leading to the establishment and maintenance of tissue-specific, epigenetic pattern is still poorly understood. In this work we propose, and investigate in silico, a possible experimental strategy to illuminate the interplay between 3D chromatin structure and epigenetic dynamics. We consider a set-up where a reconstituted chromatin fibre is stretched at its two ends (e.g., by laser tweezers), while epigenetic enzymes (writers) and chromatin-binding proteins (readers) are flooded into the system. We show that, by tuning the stretching force and the binding affinity of the readers for chromatin, the fibre undergoes a sharp transition between a stretched, epigenetically disordered, state and a crumpled, epigenetically coherent, one. We further investigate the case in which a knot is tied along the chromatin fibre, and find that the knotted segment enhances local epigenetic order, giving rise to “epigenetic solitons” which travel and diffuse along chromatin. Our results point to an intriguing coupling between 3D chromatin topology and epigenetic dynamics, which may be investigated via single molecule experiments.