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The three-dimensional structure of chromatin has emerged as an important feature of eukaryotic gene regulation. Recent technological advances in DNA sequencing-based assays have revealed locus- and chromatin state-specific structural patterns at the length scale of a few nucleosomes (~1 kb). However, interpreting these data sets remains challenging. Radiation-induced correlated cleavage of chromatin (RICC-seq) is one such chromatin structure assay that maps DNA-DNA-contacts at base pair resolution by sequencing single-stranded DNA fragments released from irradiated cells. Here, we develop a flexible modeling and simulation framework to enable the interpretation of RICC-seq data in terms of oligonucleosome structure ensembles. Nucleosomes are modeled as rigid bodies with excluded volume and adjustable DNA wrapping, connected by linker DNA modeled as a worm-like chain. We validate the model's parameters against cryo-electron microscopy and sedimentation data. Our results show that RICC-seq is sensitive to nucleosome spacing, nucleosomal DNA wrapping, and the strength of inter-nucleosome interactions. We show that nucleosome repeat lengths consistent with orthogonal assays can be extracted from experimental RICC-seq data using a 1D convolutional neural net trained on RICC-seq signal predicted from simulated ensembles. We thus provide a suite of analysis tools that add quantitative structural interpretability to RICC-seq experiments.

In eukaryotes, genomic DNA is packaged into chromatin in the nucleus. The accessibility of DNA is dependent on the chromatin structure and dynamics, which essentially control DNA-related processes, including transcription, DNA replication, and repair. All of the factors that affect the structure and dynamics of nucleosomes, the nucleosome–nucleosome interaction interfaces, and the binding of linker histones or other chromatin-binding proteins need to be considered to understand the organization and function of chromatin fibers. In this review, we provide a summary of recent progress on the structure of chromatin fibers in vitro and in the nucleus, highlight studies on the dynamic regulation of chromatin fibers, and discuss their related biological functions and abnormal organization in diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Abstract Background Posttranslational modifications of core histones are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication. Results Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using a CENP-A acetyl or unacetylatable mimic (K124Q and K124A, respectively) reveal alterations in CENP-C levels and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing. Purification of native CENP-A proteins followed by mass spectrometry analysis reveals that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early S and mid-S phases. Finally, we provide evidence implicating the histone acetyltransferase (HAT) p300 in this cycle. Conclusions Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome contribute to the binding of key kinetochore proteins, the integrity of mitosis, and centromeric replication. These data support the paradigm that modifications in histone variants can influence key biological processes.

We developed a microfluidic device with microstructures that can moor the ends of chromatin fibers obtained by unfolding single intact chromosomes. This mooring involves trapping both ends of the chromosome with antibody-conjugated microspheres and placing each microsphere on the microstructure. In principle, the device is structured in such a way that mooring of chromatin fibers, i.e., partially unfolded chromosomes, is achieved under uniform tension, which is necessary to obtain an accurate distribution of folded/unfolded regions and fluorescence probes on the chromatin fibers. In addition, changes in the distribution of chromatin folding structures were observed by controlling the solution conditions, and specific histone chemical modification regions were visualized by immunofluorescence staining of chromatin fibers in the elongated form with uniform tension. This method is advantageous for the epigenetic analysis of non-fragmented natural chromatin fibers. • A microfluidic device that can isolate and stretch intact chromosomes was developed. • Stretched chromosomes were moored under uniform tension with microstructures. • Non-uniform partial unfolding of chromosomes along chromatin fibers was observed. • Positional relationship between chromatin folding and modified histone was observed. [ABSTRACT FROM AUTHOR]

The organization and dynamics of chromatin fiber play crucial roles in regulating DNA accessibility for gene expression. Here we combine cryoelectron tomography (cryo-ET), sub-volume averaging, and 3D segmentation to visualize the in vitro and in vivo chromatin fibers folding by linker histone. We discover that an increased nucleosome repeat length and prolonged fiber length do not change the two-start helical architecture in reconstituted chromatin of homogeneous composition. Additionally, an isolated chromatin fiber with heterogeneous composition was observed, which includes short-range regions compatible with two-start helix. In vivo , sub-volume averaging reveals similar subunits of two-start helical architecture in transcriptionally inactive chromatin in frog erythrocyte nuclei. Strikingly, unambiguous DNA trajectories that displayed a zigzag pattern universally between alternate N/N+2 nucleosomes were further determined by cryo-ET with voltage phase plate. Therefore, these structural similarities suggest a general folding mode of chromatin induced by linker histone, and heterogeneous compositions mainly affect local conformation rather than changing the overall architecture. [Display omitted] • Chromatin fiber both in vitro and in vivo shows a general folding mode of two-start zigzag • Unambiguous DNA trajectories indicate a universal zigzag pattern • The heterogeneity of nucleosomes mainly affects local conformation of chromatin Li et al. reveal a universal two-start zigzag architecture and a general folding mode of chromatin fiber, in which the DNA trajectories display an unambiguous zigzag pattern both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The spatial organization of nucleosomes in 30-nm fibers remains unknown in detail. To tackle this problem, we analyzed all stereochemically possible configurations of two-start chromatin fibers with DNA linkers L = 10-70 bp (nucleosome repeat length NRL = 157-217 bp). In our model, the energy of a fiber is a sum of the elastic energy of the linker DNA, steric repulsion, electrostatics, and the H4 tail-acidic patch interaction between two stacked nucleosomes. We found two families of energetically feasible conformations of the fibers—one observed earlier, and the other novel. The fibers from the two families are characterized by different DNA linking numbers—that is, they are topologically different. Remarkably, the optimal geometry of a fiber and its topology depend on the linker length: the fibers with linkers L = 10n and 10n + 5 bp have DNA linking numbers per nucleosome DLk >>-1.5 and -1.0, respectively. In other words, the level of DNA supercoiling is directly related to the length of the inter-nucleosome linker in the chromatin fiber (and therefore, to NRL). We hypothesize that this topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. A genome-wide analysis of the NRL distribution in active and silent yeast genes yielded results consistent with this assumption.

The accessibility of genomic DNA, as a key determinant of gene-related processes, is dependent on the packing density and structural dynamics of chromatin fiber. However, due to the highly dynamic and heterogeneous properties of chromatin fiber, it is technically challenging to study these properties of chromatin. Here, we report a strategy for dissecting the dynamics of chromatin fibers based on single-molecule magnetic tweezers. Using magnetic tweezers, we can manipulate the chromatin fiber and trace its extension during the folding and unfolding process under tension to investigate the dynamic structural transitions at single-molecule level. The highly accurate and reliable in vitro single-molecule strategy provides a new research platform to dissect the structural dynamics of chromatin fiber and its regulation by different epigenetic factors during gene expression. [ABSTRACT FROM AUTHOR]

Fragile sites (FSs) in plants have been described for species like Lolium and other grasses. Whereas in humans FSs were shown to be involved in genome instabilities; the consequences of FSs expression in plants are not known yet. To evaluate whether FSs cause karyotype instabilities, we assessed the frequency of micronuclei and lagging chromosomes in meristematic cells, the stability of the DNA content, and the occurrence of neocentromeres in the presumed chromosomal fragments of Lolium perenne, Lolium multiflorum, Festuca arrundinacea, and two Festulolium hybrids. The cell cycle analysis along with flow cytometric genome size measurements showed high stability in all genomes evaluated. Neocentromeric activity was neither observed in the presumed fragments nor in any other chromosomal region, then this is not the mechanism responsible by the stability. However, Fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe in combination with YOYO staining of metaphasic chromosomes showed that many extended nucleolus organizing region (NOR) form very thin YOYO-positive chromatin fibers connecting the acentric 'fragment' with the centromere-containing chromosome region. The obtained data indicate that the expression of FSs does not result in genome instabilities or neocentromere formation. The FS-containing 45S rDNA carrying chromatin fibers undergo a cell cycle and gene activity-dependent dynamic decondensation process. [ABSTRACT FROM AUTHOR]