Organization Of Chromatin Fiber Research Articles

Computational Modeling of Chromatin Fiber to Characterize Its Organization Using Angle-Resolved Scattering of Circularly Polarized Light.

Understanding the structural organization of chromatin is essential to comprehend the gene functions. The chromatin organization changes in the cell cycle, and it conforms to various compaction levels. We investigated a chromatin solenoid model with nucleosomes shaped as cylindrical units arranged in a helical array. The solenoid with spherical-shaped nucleosomes was also modeled. The changes in chiral structural parameters of solenoid induced different compaction levels of chromatin fiber. We calculated the angle-resolved scattering of circularly polarized light to probe the changes in the organization of chromatin fiber in response to the changes in its chiral parameters. The electromagnetic scattering calculations were performed using discrete dipole approximation (DDA). In the chromatin structure, nucleosomes have internal interactions that affect chromatin compaction. The merit of performing computations with DDA is that it takes into account the internal interactions. We demonstrated sensitivity of the scattering signal’s angular behavior to the changes in these chiral parameters: pitch, radius, the handedness of solenoid, number of solenoid turns, the orientation of solenoid, the orientation of nucleosomes, number of nucleosomes, and shape of nucleosomes. These scattering calculations can potentially benefit applying a label-free polarized-light-based approach to characterize chromatin DNA and chiral polymers at the nanoscale level.

Chromosome inner structure investigation by electron tomography and electron diffraction in a transmission electron microscope

Our understanding of the inner structure of metaphase chromosomes remains inconclusive despite intensive studies using multiple imaging techniques. Transmission electron microscopy has been extensively used to visualize chromosome ultrastructure. This review summarizes recent results obtained using two transmission electron microscopy-based techniques: electron tomography and electron diffraction. Electron tomography allows advanced three-dimensional imaging of chromosomes, while electron diffraction detects the presence of periodic structures within chromosomes. The combination of these two techniques provides results contributing to the understanding of local structural organization of chromatin fibers within chromosomes.

Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy.

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.

Orientation Dependence of Inter-NCP Interaction: Insights into the Behavior of Liquid Crystal Phase and Chromatin Fiber Organization.

We report equilibrium and nonequilibrium molecular dynamics (MD) simulations of two nucleosome core particles (NCPs) stacked with their dyad axes oriented in parallel or antiparallel fashion. From the equilibrium trajectories, we determine the bridging behavior of different histone tails and observe that different sets of histone tails play important roles in the two orientations in stabilizing the NCP stack. While the H4 and H2A tails play important intermediary roles in the parallel stack, the H3 and H2B tails are important in the antiparallel stack. We use steered MD simulations to unstack the two NCPs and find a stark difference in their unstacking pathways. While the average rupture force was found to be higher for the parallel stack, the work done for complete unstacking was similar for both orientations. We use Jarzynski equality to determine the PMF profiles along the unstacking pathway, relate our findings to the behavior of NCP mesophases, and derive insights into the enigmatic nucleosomal organization in the chromatin fiber.

Capturing Structural Heterogeneity in Chromatin Fibers

Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome–nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome–nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context.

Quantitative analysis of chromatin accessibility in mouse embryonic fibroblasts

Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility.

Structure and Epigenetic Regulation of Chromatin Fibers.

In eukaryotes, genomic DNA is hierarchically packaged by histones into chromatin on several levels to fit inside the nucleus. As a central-level structure between nucleosomal arrays and higher-order chromatin organizations, the 30-nm chromatin fiber and its dynamics play a crucial role in gene regulation. However, despite considerable efforts over the past three decades, the fundamental structure and its dynamic regulation of chromatin fibers still remain as a big challenge in molecular biology. Here, we mainly summarize the most recent progress in elucidating the structure of the 30-nm chromatin fiber in vitro and epigenetic regulation of chromatin fibers by chromatin factors, particularly histone variants. In addition, we also discuss recent studies in unraveling the three-dimensional organization of chromatin fibers in situ by genomic approaches and electron microscopy.

FACT Remodels the Tetranucleosomal Unit of Chromatin Fibers for Gene Transcription

In eukaryotes, the packaging of genomic DNA into chromatin plays a critical role in gene regulation. However, the dynamic organization of chromatin fibers and its regulatory mechanisms remain poorly understood. Using single-molecule force spectroscopy, we reveal that the tetranucleosomes-on-a-string appears as a stable secondary structure during hierarchical organization of chromatin fibers. The stability of the tetranucleosomal unit is attenuated by histone chaperone FACT (facilitates chromatin transcription) invitro. Consistent with invitro observations, our genome-wide analysis further shows that FACT facilitates gene transcription by destabilizing the tetranucleosomal unit of chromatin fibers in yeast. Additionally, we found that the linker histone H1 not only enhances the stability but also facilitates the folding and unfolding kinetics of the outer nucleosomal wrap. Our study demonstrates that the tetranucleosome is a regulatory structural unit of chromatin fibers beyond the nucleosome and provides crucial mechanistic insights into the structure and dynamics of chromatin fibers during gene transcription.

Insights from All-Atom Molecular Dynamics Simulations of 40 Nucleosome Chromatin Fiber

Gene expression is regulated, in part, by the organization of chromatin fiber, which is the next hierarchical level of chromatin compaction beyond the nucleosome. Due to the large size of the fiber - millions of atoms - computational studies investigating its organization have typically used coarse-grain simulations. Such simulations use customized, relatively unproven, force fields, and fail to elicit the finer details of the atom level structure. We use multiscale all-atom simulations of 40 nucleosome (over 1 million atom) chromatin fiber to study its structure and response to modifications such as post-translational modifications implicated in chromatin remodeling. The multiscale method used, Hierarchical Charge Partitioning (HCP), exploits the natural organization of biomolecules (atoms, groups, chains, and complexes), to speedup implicit solvent simulations of the fiber by over 2 orders of magnitude. The method uses the proven Amber force field to compute interactions between nearby atoms, while approximating interactions with distant components by a smaller number of charges that optimally reproduce the low order multipole moments of these components. We use the novel technique to gain insight into the organization of the more flexible regions of the fiber, such as linker DNA and histone tails.

Chromatin structure in situ: the contribution of DNA ultrastructural cytochemistry

Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin.

Elevated embryonic expression of chromatin architectural protein HMGN5 alters higher order chromatin organization and leads to postnatal cardiac malfunction

HMGN5 is a chromatin architectural protein that specifically binds to nucleosomes, unfolds chromatin and affects transcription. We report that HMGN5 is highly and ubiquitously expressed at preimplantation stages of embryonic development but is significantly down regulated in the embryo during implantation. To assess the importance of developmentally regulated HMGN5 expression, we generated conditional transgenic mice and induced exogenous HMGN5 expression either in the whole embryo or only in the embryonic heart. We found that upregulation of HMGN5 did not alter embryonic development and the mice were born normal. However, transgenic animals remained small in size and died within 2–3 months after birth. Pathological examination revealed significant alteration in the nuclear size and chromatin architecture of cardiomyocytes evident by reorganization of chromocenters. In addition, biochemical analysis showed upregulation of genes associated with cardiac stress. MRI tests indicated severe cardiac malfunction and hypertrophy. Our studies provide a direct link between global structural organization of chromatin fiber in the heart and a phenotypic outcome evident at the level of an entire organism.

Modeling Studies of Chromatin Fiber Structure as a Function of DNA Linker Length

Chromatin fibers encountered in various species and tissues are characterized by different nucleosome repeat lengths (NRLs) of the linker DNA connecting the nucleosomes. While single cellular organisms and rapidly growing cells with high protein production have short NRL ranging from 160 to 189 bp, mature cells usually have longer NRLs ranging between 190 and 220 bp. Recently, various experimental studies have examined the effect of NRL on the internal organization of chromatin fiber. Here, we investigate by mesoscale modeling of oligonucleosomes the folding patterns for different NRL, with and without linker histone (LH), under typical monovalent salt conditions using both one-start solenoid and two-start zigzag starting configurations. We find that short to medium NRL chromatin fibers (173 to 209 bp) with LH condense into zigzag structures and that solenoid-like features are viable only for longer NRLs (226 bp). We suggest that medium NRLs are more advantageous for packing and various levels of chromatin compaction throughout the cell cycle than their shortest and longest brethren; the former (short NRLs) fold into narrow fibers, while the latter (long NRLs) arrays do not easily lead to high packing ratios due to possible linker DNA bending. Moreover, we show that the LH has a small effect on the condensation of short-NRL arrays but has an important condensation effect on medium-NRL arrays, which have linker lengths similar to the LH lengths. Finally, we suggest that the medium-NRL species, with densely packed fiber arrangements, may be advantageous for epigenetic control because their histone tail modifications can have a greater effect compared to other fibers due to their more extensive nucleosome interaction network.

Exploring the Conformational Space of Chromatin Fibers and Their Stability by Numerical Dynamic Phase Diagrams

The three-dimensional structure of chromatin affects DNA accessibility and is therefore a key regulator of gene expression. However, the path of the DNA between consecutive nucleosomes, and the resulting chromatin fiber organization remain controversial. The conformational space available for the folding of the nucleosome chain has been analytically described by phase diagrams with a two-angle model, which describes the chain trajectory by a DNA entry-exit angle at the nucleosome and a torsion angle between consecutive nucleosomes. Here, a novel type of numerical phase diagrams is introduced that relates the geometric phase space to the energy associated with a given chromatin conformation. The resulting phase diagrams revealed differences in the energy landscape that reflect the probability of a given conformation to form in thermal equilibrium. Furthermore, we investigated the effects of entropy and additional degrees of freedom in the dynamic phase diagrams by performing Monte Carlo simulations of the initial chain trajectories. Using our approach, we were able to demonstrate that conformations that initially were geometrically impossible could evolve into energetically favorable states in thermal equilibrium due to DNA bending and torsion. In addition, dynamic phase diagrams were applied to identify chromatin fibers that reflect certain experimentally determined features.

Mapping in Vivo Chromatin Interactions in Yeast Suggests an Extended Chromatin Fiber with Regional Variation in Compaction

The higher order arrangement of nucleosomes and the level of compaction of the chromatin fiber play important roles in the control of gene expression and other genomic activities. Analysis of chromatin in vitro has suggested that under near physiological conditions chromatin fibers can become highly compact and that the level of compaction can be modulated by histone modifications. However, less is known about the organization of chromatin fibers in living cells. Here, we combine chromosome conformation capture (3C) data with distance measurements and polymer modeling to determine the in vivo mass density of a transcriptionally active 95-kb GC-rich domain on chromosome III of the yeast Saccharomyces cerevisiae. In contrast to previous reports, we find that yeast does not form a compact fiber but that chromatin is extended with a mass per unit length that is consistent with a rather loose arrangement of nucleosomes. Analysis of 3C data from a neighboring AT-rich chromosomal domain indicates that chromatin in this domain is more compact, but that mass density is still well below that of a canonical 30 nm fiber. Our approach should be widely applicable to scale 3C data to real spatial dimensions, which will facilitate the quantification of the effects of chromatin modifications and transcription on chromatin fiber organization.

Structural families of genomic microsatellites

We present an analysis of tandem repeats of short sequence motifs (microsatellites) in twelve eukaryotes for which a large part of the genome has been sequenced and assembled. The pattern of motif abundance varies significantly in different species, but it is very similar in different chromosomes of the same species. The most abundant repeats can be classified in two main families. The first family has a rigid conformation, with purines in one strand and pyrimidines in the complementary strand, mainly A n /T n and (AG) n /(CT) n . The second family has alternating, flexible sequences, such as (AT) n , (AC) n and related sequences. In the pluricellular organisms the relative frequency of both families is rather constant. These observations indicate that microsatellites have structural information and may be involved in the organization of chromatin fibers and in chromosome architecture in general. An additional intriguing finding is the absence of microsatellites with sequences which appear to be forbidden, such as (AATT) n . Additional tables and references are given as supplementary material.

Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes.

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 microm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like "knobby fiber." These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher-order structures by AFM and other high-resolution microscopy.

A multiple technical approach to the study of apoptotic cell micronuclei.

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.

Three-dimensional organization of chromatin fibers in situ examined by EM tomography

Electron tomography is being used to understand the 3D organization of chromatin in situ. As demonstrated previously, the nuclei of Patiria miniata (starfish) sperm contain particularly well-defined chromatin fibers. These studies are being extended through the analysis of 3D reconstructions of material embedded at low temperature in Lowicryl K11M and contrasted with osmium ammine-B, which preferentially stains nucleic acids. Tilt series of sections were recorded at 150KV, over an angular range of +/−75° and tilt increment of 2.5° using a Philips EM430. Image data were collected directly using a 1024x1024 CCD array with 2x2 binning to give a final pixel size of 1.3nm. Gold beads deposited on the sections were used for alignment, and reconstruction was by weighted back projection. Six volumes totalling 0.48um and containing numerous chromatin fibers have been examined utilizing Voxel View (Vital Images, Fairfield Iowa) software running on a Silicon Graphics Iris 4D workstation.

Ultrastructural analysis of radiation induced chromosome breaks and rearrangements.

Chinese Hamster chromosomes R-banded in vitro were gamma-irradiated and chromatid breaks and rearrangements examined by electron microscopy employing whole-mounting tech nique. Breaks were preferentially located at the point of transition between G- and R-bands where the chromosome showed an average diameter 71.65% of the wide condensed R-bands. This result was similar to the average diameter of narrow G-bands. Three chromosomes which were thin sectioned presented their broken terminal end organized as a coil constituted by two 23 nm wide chromatin fibers coiling together. Coils diameter was 43.70% of the mean chromatid diameter. The border of damage-breakage was analyzed in whole-mounted chromosomes where breaks were photoinduced in BrdU-substituted DNA. Measurements of the angle of the sharp border of damage with respect to the chromatid axis showed a tendency to be more perpendicular as condensation progressed. These results clearly correlate with the several levels of chromatin fiber organization of the metaphase chromosome.

Involvement of the domains of histones H1 and H5 in the structural organization of soluble chromatin

We have studied in reconstitution experiments the conditions under which peptides derived from histones H1 and H5 are bound in chromatin and to what extent they are involved in the organization of chromatin fibers. The fragments of rat liver histone H1 (rH1) and chicken erythrocytes H1 (cH1) and H5 (cH5) used were the globular domains (rG-H1, cG-H1, cG-H5), the globular domain and the N-terminal tail (rCT-N), about half of the globular domain and the C-terminal tail (rNBS-C) and the C-terminal tail (rCT-C). Fragments containing the C-terminal tail (rNBS-C and rCT-C) dissociate from H1-depleted rat liver chromatin at 300 mM-NaCl and above (similar to uncleaved H1) and fragments lacking the C-terminal tail (rG-H1 and rCT-N) dissociate between 100 and 200 mM-NaCl. This suggests that at putative physiological ionic strengths the binding of rH1 is dominated by its C-terminal tail, whereas the globular region and the N-terminal tail might only be loosely bound or not bound at all and by this modulate chromatin structure. The globular domain of cH5 binds more tightly than that of the chicken and rat H1 and is only partially released at 200 mM. Since in the transcriptionally silent erythrocytes of birds H5 replaces H1 to a large extent, we suggest that the globular domain of H1 serves as a temporary seal and that of H5 as a permanent seal of the nucleosome. All the H1 and H5 peptides tested condensed and precipitated chromatin and H1-depleted chromatin: rNBS-C and rCT-C at lower peptide per nucleosome ratios than rG-H1, cG-H1 and rCT-N. At about one peptide per nucleosome none of the H1 fragments induced condensation similar to that of native chromatin. At a peptide per nucleosome ratio close to the point of precipitation, all H1 fragments, but not poly-L-lysine, induced similar compact forms which were fiberlike, although more irregular than the compact fibers of native chromatin. These reconstitution experiments suggest that both halves of H1 as well as the globular domain by itself are involved and capable in forming higher-order chromatin structures. Details of these structures are not known.