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Understanding how the genome is structurally organized as chromatin is essential for understanding its function. Here, we review recent developments that allowed the readdressing of old questions regarding the primary level of chromatin structure, the arrangement of nucleosomes along the DNA and the folding of the nucleosome fiber in nuclear space. In contrast to earlier views of nucleosome arrays as uniformly regular and folded, recent findings reveal heterogeneous array organization and diverse modes of folding. Local structure variations reflect a continuum of functional states characterized by differences in post-translational histone modifications, associated chromatin-interacting proteins and nucleosome-remodeling enzymes. Technological advances have led to new insights into genome-wide arrangements of nucleosomes along the DNA and the folding of the chromosome fiber in nuclear space, revealing unexpected diversity.

The organization and regulation of genomic DNA as nuclear chromatin is necessary for proper DNA function inside living eukaryotic cells. While this has been extensively explored, no true consensus is currently reached regarding the exact mechanism of chromatin organization. The traditional view has assumed that the DNA is packaged into a hierarchy of structures inside the nucleus based on the regular 30-nm chromatin fiber. This is currently being challenged by the fluid-like model of the chromatin which views the chromatin as a dynamic structure based on the irregular 10-nm fiber. In this review, we focus on the recent progress in chromatin structure elucidation highlighting the paradigm shift in chromatin folding mechanism from the classical textbook perspective of the regularly folded chromatin to the more dynamic fluid-like perspective.

Nucleosomes cover eukaryotic genomes like beads on a string and play a central role in regulating genome function. Isolated strings of nucleosomes have the potential to compact and form higher order chromatin structures, such as the well-characterized 30-nm fiber. However, despite tremendous advances in observing chromatin fibers in situ it has not been possible to confirm that regularly ordered fibers represent a prevalent structural level in the folding of chromosomes. Instead, it appears that folding at a larger scale than the nucleosome involves a variety of random structures with fractal characteristics. Nevertheless, recent progress provides evidence for the existence of structural motifs in chromatin fibers, potentially localized to strategic sites in the genome. Here we review the current understanding of chromatin fiber folding and the emerging roles that oligonucleosomal motifs play in the regulation of genome function.

In the nuclei of eukaryotic cells, the genetic information is organized at several levels. First, the DNA is wound around the histone proteins, to form a structure termed as chromatin fiber. This fiber is then arranged into chromatin loops that can cluster together and form higher order structures. This packaging of chromatin provides on one side compaction but also functional compartmentalization. The cohesin complex is a multifunctional ring-shaped multiprotein complex that organizes the chromatin fiber to establish functional domains important for transcriptional regulation, help with DNA damage repair, and ascertain stable inheritance of the genome during cell division. Our current model for cohesin function suggests that cohesin tethers chromatin strands by topologically entrapping them within its ring. To achieve this, cohesin’s association with chromatin needs to be very precisely regulated in timing and position on the chromatin strand. Here we will review the current insight in when and where cohesin associates with chromatin and which factors regulate this. Further, we will discuss the latest insights into where and how the cohesin ring opens to embrace chromatin and also the current knowledge about the ‘exit gates’ when cohesin is released from chromatin.

International audience; The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.

Background H2A.B, the most divergent histone variant of H2A, can significantly modulate nucleosome and chromatin structures. However, the related structural details and the underlying mechanism remain elusive to date. In this work, we built atomic models of the H2A.B-containing nucleosome core particle (NCP), chromatosome, and chromatin fiber. Multiscale modeling including all-atom molecular dynamics and coarse-grained simulations were then carried out for these systems. Results It is found that sequence differences at the C-terminal tail, the docking domain, and the L2 loop, between H2A.B and H2A are directly responsible for the DNA unwrapping in the H2A.B NCP, whereas the N-terminus of H2A.B may somewhat compensate for the aforementioned unwrapping effect. The assembly of the H2A.B NCP is more difficult than that of the H2A NCP. H2A.B may also modulate the interactions of H1 with both the NCP and the linker DNA and could further affect the higher-order structure of the chromatin fiber. Conclusions The results agree with the experimental results and may shed new light on the biological function of H2A.B. Multiscale modeling may be a valuable tool for investigating structure and dynamics of the nucleosome and the chromatin induced by various histone variants.

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo., e acknowledge support from European Union’s Horizon 2020 research and innovation program (CellViewer 686637 to J.S., M.L., and M.P.C.); Ministerio de Ciencia e Innovación (grant BFU2017-86760-P [AEI/FEDER, UE] to M.P.C.), and an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya (2017 SGR 689 to M.P.C.); Centro de Excelencia Severo Ochoa (2013–2017 to M.P.C.); CERCA Programme/Generalitat de Catalunya (to M.P.C.); the Spanish Ministry of Science and Innovation to the European Molecular Biology Laboratory (EMBL) partnership (to M.P.C.); the National Natural Science Foundation of China (31971177 to M.P.C.); the Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110103001 to M.P.C.); a Linda Pechenik Montague Investigator Award (to M.L.); a University of Pennsylvania Epigenetics Pilot Award (to M.L.); the Center for Engineering and Mechanobiology (CEMB) a National Science Foundation (NSF) Science and Technology Center Pilot Award (Division of Civil, Mechanical and Manufacturing Innovation [CMMI]: 15-48571 to M.L.); NIH grants R01HD091797 and R01GM123289 (to C.t.W.); the People Program (Marie Skłodowska-Curie Actions) FP7/2007–2013 under an REA grant (608959 to M.V.N.); Juan de la Cierva-Incorporación 2017 (to M.V.N.); Grant for the recruitment of early-stage research staff FI-2020 (Operational Program of Catalonia 2014–2020 CCI 2014ES05SFOP007 of the European Social Fund to L.M.); a “la Caixa” Foundation Fellowship (ID 100010434, LCF/BQ/DR20/11790016) (to L.M.); “La Caixa-Severo Ochoa” pre-doctoral fellowship (to Á.C.-G.); and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and the co-financing of Fondo Social Europeo (2018FI_B_00637 and FSE to R.S.-P.), and a La Caixa international PhD fellowship (to F.S.).

Chromatin organization starts from a “beads-on-a string” 10 nm fiber, a basic nucleosomal structure consisting of DNA and core histones. Given its regular nucleosome array on DNA backbone where N-terminal tails of each histone are exposed on the surface of chromatin fiber, we hypothesized that chromatin can be utilized as a heterologous peptide carrier to elicit a peptide-specific immune response. The plasmid DNA containing the Widom’s clone 601 sequence and the recombinant chimeric histones containing the peptide derived from ras oncogene (G12V) were used to assemble the chromatin fiber in vitro. The immunogenicity of the assembled chromatin was tested in mice as a single vaccine component or formulated with adjuvants. G12V tagged-chromatin co-administered with adjuvants induced higher antibody responses against the G12V peptide than vaccination with adjuvant alone, while chimeric histones did not generate a significant antibody response. Interestingly, splenocytes from mice vaccinated with the G12V tagged-chromatin vaccine did not generate significant antigen-specific cytokine responses. Our studies suggest that chromatin can be utilized as an effective carrier of antigenic peptides for inducing specific antibody responses.

In mammals, genomic DNA that is roughly 2 m long is folded to fit the size of the cell nucleus that has a diameter of about 10 μm. The folding of genomic DNA is mediated via assembly of DNA-protein complex, chromatin. In addition to the reduction of genomic DNA linear dimensions, the assembly of chromatin allows to discriminate and to mark active (transcribed) and repressed (non-transcribed) genes. Consequently, epigenetic regulation of gene expression occurs at the level of DNA packaging in chromatin. Taking into account the increasing attention of scientific community toward epigenetic systems of gene regulation, it is very important to understand how DNA folding in chromatin is related to gene activity. For many years the hierarchical model of DNA folding was the most popular. It was assumed that nucleosome fiber (10-nm fiber) is folded into 30-nm fiber and further on into chromatin loops attached to a nuclear/chromosome scaffold. Recent studies have demonstrated that there is much less regularity in chromatin folding within the cell nucleus. The very existence of 30-nm chromatin fibers in living cells was questioned. On the other hand, it was found that chromosomes are partitioned into self-interacting spatial domains that restrict the area of enhancers action. Thus, TADs can be considered as structural-functional domains of the chromosomes. Here we discuss the modern view of DNA packaging within the cell nucleus in relation to the regulation of gene expression. Special attention is paid to the possible mechanisms of the chromatin fiber self-assembly into TADs. We discuss the model postulating that partitioning of the chromosome into TADs is determined by the distribution of active and inactive chromatin segments along the chromosome. This article was specially invited by the editors and represents work by leading researchers.

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

SUMMARY Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

International audience; Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

Chromatin is the epigenomic platform for diverse nuclear processes such as DNA repair, replication, transcription, telomere, and centromere function. In cancer cells, mutations in key processes result in DNA amplification, chromosome translocations, and chromothripsis, severely distorting the natural chromatin state. In normal and diseased states, dozens of chromatin effectors alter the physical integrity and dynamics of chromatin at the level of both single nucleosomes and arrays of nucleosomes folded into 3-dimensional shapes. Integrating these length scales, from the 10 nm sized nucleosome to mitotic chromosomes, whilst jostling within the crowded environment of the cell, cannot yet be achieved by a single technology. In this review, we discuss tools that have proven powerful in the investigation of nucleosome and chromatin fiber dynamics. We also provide a deeper focus into atomic force microscopy (AFM) applications that can bridge diverse length and time scales. Using time course AFM, we observe that chromatin condensation by H1.5 is dynamic, whereas using nano-indentation force spectroscopy we observe that both histone variants and nucleosome binding partners alter material properties of individual nucleosomes. Finally, we demonstrate how high-speed AFM can visualize plasmid DNA dynamics, intermittent nucleosome-nucleosome contacts, and changes in nucleosome phasing along a contiguous chromatin fiber. Altogether, the development of innovative technologies holds the promise of revealing the secret lives of nucleosomes, potentially bridging the gaps in our understanding of how chromatin works within living cells and tissues.

In this paper, a geometric model of metaphase chromosome is explained. The model is constructed according to the packing ratio and dimension of the structure from nucleosome up to chromosome. A B-DNA base pair is used to construct 200 base pairs of nucleosomes. Each chromatin fiber loop, which is the unit of repeat, has 49,200 bp. This geometry is entered in Geant4.10 Monte Carlo simulation toolkit and can be extended to the whole metaphase chromosomes and any application in which a DNA geometrical model is needed. The chromosome base pairs, chromosome length, and relative length of chromosomes are calculated. The calculated relative length is compared to the relative length of human chromosomes.

The cohesin complex has evolved as major player for structuring the chromatin fiber with roles in chromosome segregation, chromatin architecture, DNA damage repair and gene regulation. This article summarizes the latest discoveries and models for cohesin function and also reviews implications of the cohesin complex in developmental syndromes and cancer.

It has long been suggested that chromatin may form a fiber with a diameter of ~30 nm that suppresses transcription. Despite nearly four decades of study, the structural nature of the 30-nm chromatin fiber and conclusive evidence of its existence in vivo remain elusive. The key support for the existence of specific 30 nm chromatin fiber structures is based on the determination of the structures of reconstituted nucleosome arrays using X-ray crystallography and single particle cryo-electron microscopy coupled with glutaraldehyde chemical cross-linking. Here we report the characterization of these nucleosome arrays in solution using analytical ultracentrifugation, nuclear magnetic resonance, and small angle X-ray scattering. We found that the physical properties of these nucleosome arrays in solution are not consistent with formation of just a few discrete structures of nucleosome arrays. In addition, we obtained a crystal of the nucleosome in complex with the globular domain of linker histone H5 that shows a new form of nucleosome packing and suggests a plausible alternative compact conformation for nucleosome arrays. Taken together, our results challenge the key evidence for the existence of a limited number of structures of reconstituted nucleosome arrays in solution by revealing that the reconstituted nucleosome arrays are actually best described as an ensemble of various conformations with a zigzagged arrangement of nucleosomes. Our finding has implications for understanding the structure and function of chromatin in vivo.

Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (∼200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy ( dSTORM) with a lateral resolution of ∼20 nm to analyze the focal nanostructure of the DSB marker histone γH2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained γH2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that γH2AX foci consisted of distinct circular subunits ("nanofoci") with a diameter of ∼45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that γH2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual γH2AX-containing nucleosomes. dSTORM-based γH2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of γH2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ∼0.6 and ∼1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.-Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.

Background DNA inside eukaryotic cells wraps around histones to form the 11nm chromatin fiber that can further fold into higher-order DNA loops, which may depend on the binding of architectural factors. Predicting how the DNA will fold given a distribution of bound factors, here viewed as a type of sequence, is currently an unsolved problem and several heterogeneous polymer models have shown that many features of the measured structure can be reproduced from simulations. However a model that determines the optimal connection between sequence and structure and that can rapidly assess the effects of varying either one is still lacking. Results Here we train a dense neural network to solve for the local folding of chromatin, connecting structure, represented as a contact map, to a sequence of bound chromatin factors. The network includes a convolutional filter that compresses the large number of bound chromatin factors into a single 1D sequence representation that is optimized for predicting structure. We also train a network to solve the inverse problem, namely given only structural information in the form of a contact map, predict the likely sequence of chromatin states that generated it. Conclusions By carrying out sensitivity analysis on both networks, we are able to highlight the importance of chromatin contexts and neighborhoods for regulating long-range contacts, along with critical alterations that affect contact formation. Our analysis shows that the networks have learned physical insights that are informative and intuitive about this complex polymer problem. Electronic supplementary material The online version of this article (10.1186/s12859-018-2286-z) contains supplementary material, which is available to authorized users.

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.

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.

How chromosomes are folded and how this folding relates to function remain fundamental questions. Answering them is rendered difficult by the stochasticity of chromatin fiber motion which inevitably results in heterogeneity of the populations analyzed. Even if single cell analyses are beginning to yield precious insights, how can we determine whether a snapshot of position is related to function of the probed locus or cell-type? Fluorescence labeling of DNA at single or multiple loci allows determination of their position relative to nuclear landmarks and to each other, enabling us to derive physical parameters of the underlying chromatin fiber. Here I review the contribution of quantitative spatial and temporal analysis of labeled DNA to our understanding of chromosome conformation in different cell types, highlighting live cell imaging techniques and large scale geometrical analysis of multiple loci in 3D.

Architectural proteins mediate interactions between distant sequences in the genome. Two well-characterized functions of architectural protein interactions include the tethering of enhancers to promoters and bringing together Polycomb-containing sites to facilitate silencing. The nature of which sequences interact genome-wide appears to be determined by the orientation of the architectural protein binding sites as well as the number and identity of architectural proteins present. Ultimately, long range chromatin interactions result in the formation of loops within the chromatin fiber. In this review, we discuss data suggesting that architectural proteins mediate long range chromatin interactions that both facilitate and hinder neighboring interactions, compartmentalizing the genome into regions of highly interacting chromatin domains.

The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ∼7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones.

Some open questions on the mechanisms underlying radiation-induced cell death were addressed by a biophysical model, focusing on DNA damage clustering and its consequences. DNA "cluster lesions" (CLs) were assumed to produce independent chromosome fragments that, if created within a micrometer-scale threshold distance (d), can lead to chromosome aberrations following mis-rejoining; in turn, certain aberrations (dicentrics, rings and large deletions) were assumed to lead to clonogenic cell death. The CL yield and d were the only adjustable parameters. The model, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations (BIANCA), provided simulated survival curves that were directly compared with experimental data on human and hamster cells exposed to photons, protons, α-particles and heavier ions including carbon and iron. d = 5 μm, independent of radiation quality, and CL yields in the range ~2-20 CLs Gy(-1) cell(-1), depending on particle type and energy, led to good agreement between simulations and data. This supports the hypothesis of a pivotal role of DNA cluster damage at sub-micrometric scale, modulated by chromosome fragment mis-rejoining at micrometric scale. To investigate the features of such critical damage, the CL yields were compared with experimental or theoretical yields of DNA fragments of different sizes, focusing on the base-pair scale (related to the so-called local clustering), the kbp scale ("regional clustering") and the Mbp scale, corresponding to chromatin loops. Interestingly, the CL yields showed better agreement with kbp fragments rather than bp fragments or Mbp fragments; this suggests that also regional clustering, in addition to other clustering levels, may play an important role, possibly due to its relationship with nucleosome organization in the chromatin fiber.

The structure of chromatin, affected by many factors from DNA linker lengths to posttranslational modifications, is crucial to the regulation of eukaryotic cells. Combined experimental and computational methods have led to new insights into its structural and dynamical features, from interactions due to the flexible core histone tails or linker histones to the physical mechanism driving the formation of chromosomal domains. Here we present a perspective of recent advances in chromatin modeling techniques at the atomic, mesoscopic, and chromosomal scales with a view toward developing multiscale computational strategies to integrate such findings. Innovative modeling methods that connect molecular to chromosomal scales are crucial for interpreting experiments and eventually deciphering the complex dynamic organization and function of chromatin in the cell.

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.

Eukaryotic chromosomes are complex polymers, which largely exceed in size most biomolecules that are usually modelled in computational studies and whose molecular interactions are to a large extent unknown. Since the folding of the chromatin fiber in the cell nucleus is tightly linked to biological function and gene expression in particular, characterizing the conformational and dynamical properties of chromosomes has become crucial in order to better understand how genes are regulated. In parallel with the development of experimental techniques allowing to measure physical contacts within chromosomes inside the cell nucleus, a large variety of physical models to study the structure and mechanisms of chromosome folding have recently emerged. Such models can be roughly divided into two classes, based on whether they adopt specific hypotheses on the interaction mechanism within chromosomes, or learn those interactions on the available experimental data using the principle of maximum entropy. All of them have played a key role in interpreting experimental data and advancing our understanding the folding principles of the chromatin fiber.

The sequence of a genome is insufficient to understand all genomic processes carried out in the cell nucleus. To achieve this, the knowledge of its three-dimensional architecture is necessary. Advances in genomic technologies and the development of new analytical methods, such as Chromosome Conformation Capture (3C) and its derivatives, provide unprecedented insights in the spatial organization of genomes. Here we present TADbit, a computational framework to analyze and model the chromatin fiber in three dimensions. Our package takes as input the sequencing reads of 3C-based experiments and performs the following main tasks: (i) pre-process the reads, (ii) map the reads to a reference genome, (iii) filter and normalize the interaction data, (iv) analyze the resulting interaction matrices, (v) build 3D models of selected genomic domains, and (vi) analyze the resulting models to characterize their structural properties. To illustrate the use of TADbit, we automatically modeled 50 genomic domains from the fly genome revealing differential structural features of the previously defined chromatin colors, establishing a link between the conformation of the genome and the local chromatin composition. TADbit provides three-dimensional models built from 3C-based experiments, which are ready for visualization and for characterizing their relation to gene expression and epigenetic states. TADbit is an open-source Python library available for download from https://github.com/3DGenomes/tadbit. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement 609989, the Spanish Ministry of Economy and Competitiveness (BFU2013-47736-P) and the Human Frontiers Science Program (RGP0044). We acknowledge support of the CERCA Programme / Generalitat de Catalunya and the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017', SEV-2012-0208 to the CRG.

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 A 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.

Post-translational modifications (PTMs) of core histones have studied for over 2 decades, and 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. 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 an acetyl or unacetylatable mimic (CENP-A 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, with the permanently acetylated form replicating centromeres early, and the unacetylable form replicating centromeres late. Purification of native CENP-A proteins followed by mass spectrometry analysis reveal that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early and mid-S phase. Finally, we provide evidence that the HAT p300 is involved in this cycle. Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome can influence the binding of key kinetochore proteins, the integrity of mitosis and centromeric replication. These data support the emerging paradigm that core modifications in histone variant nucleosomes transduce defined changes to key biological processes.

Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure—a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions., Author summary The fate of a cell is not just decided by the genetic code but also by the nature of the 3D organization of the protein-bound DNA, known as chromatin. Chromatin packaging is believed to be in a hierarchical manner, and one of the crucial stages in the packaging is argued to be having a zig-zag structure with specific width of 30 nm. However, most of the recent experiments failed to find any zig-zag-like ordered arrangement of chromatin in living cells. In this work, we address this puzzle, and argue that any regular, ordered, packaging of chromatin is unviable given that certain types of proteins can bind and bend the chromosomal DNA.

The DNA of metazoan somatic cells is packaged into a compact nucleoprotein complex termed chromatin (Van Holde 1988; Wolffe 1998). Chromatin fiber is comprised of highly conserved repetitive units (nucleosomes) that contain an octamer of four core histones and 145–147 base pairs (bp) of DNA wrapped around the octamer in 1.65 turns of a left-handed superhelix (Luger et al. 1997). Nucleosomes are assembled in vivo in an ATP-dependent fashion through a concerted and sequential action of core histone chaperones and motor proteins that belong to the Snf2 family of DNA-dependent ATPases (Gorbalenya and Koonin 1993; Eisen et al. 1995; Mello and Almouzni 2001; Akey and Luger 2003; Haushalter and Kadonaga 2003; Piatti et al. 2011; Torigoe et al. 2013). For instance, Drosophila ACF/CHRAC can mediate chromatin assembly in conjunction with histone chaperone NAP-1 (Varga-Weisz et al. 1997; Ito et al. 1999). Other known ATP-dependent chromatin assembly factors include RSF, CHD1, ATRX, and ToRC/NoRC (LeRoy et al. 1998, 2000; Lusser et al. 2005; Lewis et al. 2010; Emelyanov et al. 2012). The most abundant chromatin component in male germline cells is protamines—small positively charged arginine- and cysteine-rich proteins (Balhorn 2007). During spermiogenesis, protamines replace 85%–95% of DNA-bound histones in the nucleus to achieve a higher density of sperm nuclear DNA (Ward and Coffey 1991; Rathke et al. 2014). Crystalline-like sperm chromatin structure is sixfold more compact than metaphase chromosomes and renders sperm DNA enzymatically inert (Balhorn 1982). At fertilization, the oocyte remodels the condensed sperm chromatin into a transcriptionally competent chromatin of the male pronucleus (McLay and Clarke 2003). During, this process, protamines are expelled and replaced with oocyte-supplied histones, which are then organized into nucleosomes. Sperm chromatin remodeling (SCR) is controlled by biochemical activities in the early oocyte, but components of these activities remain largely unknown (McLay and Clarke 2003). However various protein factors have been implicated in SCR, including core histone chaperones from Xenopus (Nucleoplasmin, N1, HIRA, and TAF-I) and Drosophila (NAP-1, p22, DF31, HIRA, and Yemanuclein) (Dilworth et al. 1987; Philpott and Leno 1992; Kawasaki et al. 1994; Crevel and Cotterill 1995; Ito et al. 1996a; Matsumoto et al. 1999; Loppin et al. 2001; Ray-Gallet et al. 2002; Orsi et al. 2013). In mammals, members of nucleoplasmin/nucleophosmin family proteins (NPM1–3) function in sperm chromatin decondensation in vitro (Okuwaki et al. 2012). In addition, Npm2 knockout female mice exhibit fertility defects consistent with a role of NPM2 in nuclear and nucleolar chromatin organization (Burns et al. 2003). It was also suggested that sperm chromatin decondensation is ATP-dependent (Wright and Longo 1988). Drosophila melanogaster sperm cells contain two major protamines (A and B) encoded by male-specific transcripts Mst35Ba and Mst35Bb, respectively (Ashburner et al. 1999; Jayaramaiah Raja and Renkawitz-Pohl 2005). The Drosophila maternal effect mutant sesame (ssm) prevents male pronucleus formation (Loppin et al. 2001). ssm encodes the histone variant H3.3-specific chaperone HIRA (Tagami et al. 2004), postulated to be required for replication-independent deposition of histones in the male pronucleus during sperm decondensation (Loppin et al. 2005; Bonnefoy et al. 2007). In eggs from homozygous ssm females, maternal histones are not deposited in the chromatin of male pronuclei, preventing normal mitosis and resulting in the development of gynogenetic haploid embryos and embryonic stage lethality. A similar phenotype is observed in null mutants of the gene encoding ATP-dependent chromatin assembly factor CHD1 (Konev et al. 2007). Thus, CHD1 and HIRA act cooperatively and are required for nucleosome assembly during SCR. Intriguingly, protamines are efficiently expelled from the DNA of nascent male pronuclei in Chd1 and ssm eggs (Jayaramaiah Raja and Renkawitz-Pohl 2005; Konev et al. 2007), suggesting that protamine removal and histone deposition are functionally distinct steps. In this study, we used a biochemical approach to identify specific protein components of the Drosophila egg machinery that promote the dissociation of protamine–DNA complexes of sperm chromatin. These factors turn out to be two known core histone chaperones (NAP-1 and NLP), a homolog of mammalian nucleophosmin, and a novel Drosophila histone chaperone (TAP/p32). These putative “protamine chaperones” facilitate SCR independently of CHD1 and HIRA, which mediate nucleosome assembly in nascent male pronuclei. Of note, TAP/p32 is specifically required to expel Drosophila protamine B from sperm chromatin in vitro, whereas NAP-1, NLP, and nucleophosmin share roles in removal of protamine A. We also provide in vivo evidence that TAP/p32 functions in Drosophila egg SCR. In conclusion, we characterized protein factors that mediate the first obligatory step of SCR (protamine dissociation) and reconstituted the complete SCR reaction (reorganization of protamine-containing sperm chromatin into core histone-containing nucleosome arrays) in a purified defined system in vitro.

Background: Ionizing radiation induces a plethora of DNA damage including double-strand breaks (DSB) that may trigger a series of events such as transcription, DNA repair and alteration in the conformation of chromatin structure in human cells. We have made an attempt to study the conformational changes in chromatin fibers in irradiated human peripheral blood mononuclear cells (PBMC) using Dynamic Light Scattering (DLS) as a new tool.Venous blood samples were collected from 10 random, healthy individuals with written informed consent, approved by institutional ethics committee. PBMC were separated from blood, irradiated with different doses of gamma radiation from 0.25-1.0 Gy. Native chromatin was isolated from irradiated PBMC and changes in the hydrodynamic diameter of the chromatin fiber were measured using DLS. Both dose response and time kinetics was studied in order to see the chromatin changes. Radiation-induced DNA double-strand breaks were measured using gamma-H2AX (histone 2A member X) as a biomarker using flow cytometry and foci were visualized in confocal microscopy.A significant alteration in hydrodynamic diameter of the chromatin fiber was observed at lower doses (0.25 and 0.50 Gy), whereas at higher dose (1.0 Gy), the size of the chromatin fiber was comparable to unirradiated control. Among the 10 individuals studied, five individuals showed significant increase (p ≤ 0.002) in hydrodynamic size at 0.25 Gy whereas four individuals showed significant decrease (p ≤ 0.009) at 0.25 Gy. One individual did not show any significant difference as compared to control. However, dose-dependent increase in gamma-H2AX fluorescence signals as well as foci number was observed. Increased fragmentation of chromatin fiber was also observed using Atomic Force Microscopy at higher doses.Radiation-induced DNA damage response can lead to individual specific conformational changes in chromatin structure at lower doses (0.25 Gy and 0.50 Gy) which can be detected using dynamic light scattering method in resting human PBMC.

Several hierarchical levels of DNA packaging are believed to exist in chromatin, starting from a 10-nm chromatin fiber that is further packed into a 30-nm fiber. Transitions between the 30-nm and 10-nm fibers are thought to be essential for the control of chromatin transcriptional status. However, recent studies demonstrate that in the nuclei, DNA is packed in tightly associated 10-nm fibers that are not compacted into 30-nm fibers. Additionally, the accessibility of DNA in chromatin depends on the local mobility of nucleosomes rather than on decompaction of chromosome regions. These findings argue for reconsidering the hierarchical model of chromatin packaging and some of the basic definitions of chromatin. In particular, chromatin domains should be considered as three-dimensional objects, which may include genomic regions that do not necessarily constitute a continuous domain on the DNA chain.

High resolution fiber-Fluorescence in situ hybridization (FISH) is an advanced FISH technology that can effectively bridge the resolution gap between probe hybridizing on DNA molecules and chromosomal regions. Since various types of DNA and chromatin fibers can be generated reflecting different degrees of DNA/chromatin packaging status, fiber-FISH technology has been successfully used in diverse molecular cytogenetic/cytogenomic studies. Following a brief review of this technology, including its major development and increasing applications, typical protocols to generate DNA/chromatin fiber will be described, coupled with rationales, as well as technical tips. These released DNA/chromatin fibers are suitable for an array of cytogenetic/cytogenomic analyses.

Chromatin domains formed in vivo are characterized by different types of 3D organization of interconnected nucleosomes and architectural proteins. Here, we quantitatively test a hypothesis that the similarities in the structure of chromatin fibers (which we call “structural homology”) can affect their mutual electrostatic and protein-mediated bridging interactions. For example, highly repetitive DNA sequences in heterochromatic regions can position nucleosomes so that preferred inter-nucleosomal distances are preserved on the surfaces of neighboring fibers. On the contrary, the segments of chromatin fiber formed on unrelated DNA sequences have different geometrical parameters and lack structural complementarity pivotal for stable association and cohesion. Furthermore, specific functional elements such as insulator regions, transcription start and termination sites, and replication origins are characterized by strong nucleosome ordering that might induce structure-driven iterations of chromatin fibers. We propose that shape-specific protein-bridging interactions facilitate long-range pairing of chromatin fragments, while for closely-juxtaposed fibers electrostatic forces can in addition yield fine-tuned structure-specific recognition and pairing. These pairing effects can account for some features observed for mitotic and inter-phase chromatins.

International audience; DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.

Linker histones are three domain proteins and consist of a structured (globular) domain, flanked by two likely non-structured NH2- and COOH-termini. The binding of the linker histones to the nucleosome was characterized by different methods in solution. Apparently, the globular domain interacts with the linker DNA and the nucleosome dyad, while the binding of the large and rich in lysines COOH-terminus results in "closing" the linker DNA of the nucleosome and the formation of the "stem" structure. What is the mode of binding of the linker histones within the chromatin fiber remains still elusive. Nonetheless, it is clear that linker histones are essential for both the assembly and maintenance of the condensed chromatin fiber. Interestingly, linker histones are post-translationally modified and how this affects both their binding to chromatin and functions is now beginning to emerge. In addition, linker histones are highly mobile in vivo, but not in vitro. No explanation of this finding is reported for the moment. The higher mobility of the linker histones should, however, have strong impact on their function. Linker histones plays an important role in gene expression regulation and other chromatin related process and their function is predominantly regulated by their posttranslational modifications. However, the detailed mechanism how the linker histones do function remains still not well understood despite numerous efforts. Here we will summarize and analyze the data on the linker histone binding to the nucleosome and the chromatin fiber and will discuss its functional consequences.

The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14/16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. Expression of the signature subunit ACF1 is restricted during embryonic development, but remains high in primordial germ cells. Therefore, we explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf1(7) allele leads to defective egg chambers and their elimination through apoptosis. In addition, we find a variety of unusual 16-cell cyst packaging phenotypes in the previously known Acf1(1) allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, we found that the Acf1(1) deletion - despite disruption of the Acf1 reading frame - expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis.

Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin. Using this assay, we have expressed individual H1 subtypes in the oocytes to study their effect on chromatin structure using nucleosomal repeat length (NRL) as readout. We have compared chicken differentiation-specific histone H5, Xenopus differentiation-specific xH1(0) and the somatic variant xH1A as well as the ubiquitously expressed human somatic subtypes hH1.2, hH1.3, hH1.4 and hH1.5. This shows that all subtypes, except for human H1.5, result in a saturable increase in NRL. hH1.4 results in an increase of approximately 13-20 bp as does xH1(0) and xH1A. chH5 gives rise to the same or slightly longer increase compared to hH1.4. Interestingly, both hH1.2 and hH1.3 show a less extensive increase of only 4.5-7 bp in the NRL, thus yielding the shortest increase of the studied subtypes. We show for the first time in an in vivo system lacking H1 background that ubiquitously expressed and redundant H1 subtypes that coexist in most types of cells of higher eukaryotes differ in their effects on the nucleosomal spacing in vivo. This suggests that H1 subtypes have different roles in the organization and functioning of the chromatin fiber.

Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.

How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 μm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure.

Eukaryotic gene activation requires selective unfolding of the chromatin fiber to access the DNA for processes such as DNA transcription, replication, and repair. Mutation/modification experiments of linker histone (LH) H1 suggest the importance of dynamic mechanisms for LH binding/dissociation, but the effects on chromatin's unfolding pathway remain unclear. Here we investigate the stretching response of chromatin fibers by mesoscale modeling to complement single-molecule experiments, and present various unfolding mechanisms for fibers with different nucleosome repeat lengths (NRLs) with/without LH that are fixed to their cores or bind/unbind dynamically with different affinities. Fiber softening occurs for long compared to short NRL (due to facile stacking rearrangements), dynamic compared to static LH/core binding as well as slow rather than fast dynamic LH rebinding (due to DNA stem destabilization), and low compared to high LH concentration (due to DNA stem inhibition). Heterogeneous superbead constructs—nucleosome clusters interspersed with extended fiber regions—emerge during unfolding of medium-NRL fibers and may be related to those observed experimentally. Our work suggests that fast and slow LH binding pools, present simultaneously in vivo, might act cooperatively to yield controlled fiber unfolding at low forces. Medium-NRL fibers with multiple dynamic LH pools offer both flexibility and selective DNA exposure, and may be evolutionarily suitable to regulate chromatin architecture and gene expression.

About a decade ago, the elastic properties of a single chromatin fiber and, subsequently, those of a single nucleosome started to be explored using optical and magnetic tweezers. These techniques have allowed direct measurements of several essential physical parameters of individual nucleosomes and nucleosomal arrays, including the forces responsible for the maintenance of the structure of both the chromatin fiber and the individual nucleosomes, as well as the mechanism of their unwinding under mechanical stress. Experiments on the assembly of individual chromatin fibers have illustrated the complexity of the process and the key role of certain specific components. Nevertheless a substantial disparity exists in the data reported from various experiments. Chromatin, unlike naked DNA, is a system which is extremely sensitive to environmental conditions, and studies carried out under even slightly different conditions are difficult to compare directly. In this review we summarize the available data and their impact on our knowledge of both nucleosomal structure and the dynamics of nucleosome and chromatin fiber assembly and organization.

Several different models of the linker histone (LH)-nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. According to the current paradigm, strings of nucleosomes, termed 10nm chromatin fibers, constitute the template of transcriptionally active genomic material. The majority of the genome is maintained in a silenced state through higher-order chromatin assemblies, based on the 30nm chromatin fiber, which excludes activating regulatory factors. New experimental approaches, however, including chromatin conformation capture and cryo-electron microscopy, call into question the in situ evidence for the 30nm chromatin fiber. We suggest that the organization of the genome based on 10nm chromatin fibers is sufficient to describe the complexities of nuclear organization and gene regulation.

In mammals, higher order chromatin structures are critical for downsizing the genome (packaging) so that the nucleus can be small. The adjustable density of chromatin also regulates gene expression, thus this post-genetic molecular mechanism is one of the routes by which phenotype is shaped. Phenotypes that arise without a concomitant mutation of the underlying genome are termed epigenetic phenomena. Here we discuss epigenetic phenomena from histone and DNA modification as it pertains to the dynamic regulatory processes of the circadian clock. Epigenetic phenomena certainly explain some regulatory aspects of the mammalian circadian oscillator. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Regulation of chromatin structure involves histone post-translational modifications which can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Importantly, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence based method we found that uH2B acts through a mechanism distinct from H4 tail acetylation (acH4), a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acH4 in nucleosomes resulted in synergistic inhibition of higher order chromatin structure formation, possibly a result of their distinct mode of action.

Chromatin fibers encountered in various species and tissues are characterized by different nucleosome repeat lengths (NRL) 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 base pairs (bp), mature cells usually have longer NRL 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, 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 linker histone condense into irregular zigzag structures, and that solenoid-like features are viable only for longer NRL (226 bp). We suggest that medium NRL are more advantageous for packing and various levels of chromatin compaction throughout the cell cycle than their shortest and longest brethren; the former (short NRL) fold into narrow fibers, while the latter (long NRL) arrays do not easily lead to high packing ratios due to possible linker DNA bending. Moreover, we show that the linker histone has a small effect on the condensation of short-NRL arrays but an important condensation effect on medium-NRL arrays which have linker lengths similar to the linker histone 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.