Internucleosomal Interactions Research Articles

Sequence-dependent nucleosome nanoscale structure characterized by atomic force microscopy.

DNA sequence plays an important role in the assembly of nucleosomes, and DNA motifs with high specificity to nucleosomes have been identified. At the same time, important questions such as how the DNA sequence changes DNA wrapping and how the DNA sequence contributes to the interaction between the nucleosomes remain unclear. Here, we addressed these questions by comparing nanoscale properties of nucleosomes assembled on the highest nucleosome positioning sequence, the 601 motif, and essentially random DNA sequences. We used atomic force microscopy to measure the nucleosome positions and the DNA wrapping. The studies showed that nucleosomes assemble on the nonspecific sequence without any preference for position on DNA, but they wrap the same DNA length as DNA of the same length with the 601 sequence. Experiments with longer DNA containing the 601 motif along with nonspecific DNA, capable of forming dinucleosomes, revealed that dinucleosomes assembled on hybrid sequences show a preference for positioning near each other, with one always assembling on the 601 motif. These findings point to the interaction between the nucleosomes and suggest that internucleosomal interactions may play a large role in nucleosome positioning.-Stormberg, T., Stumme-Diers, M., Lyubchenko, Y. L. Sequence-dependent nucleosome nanoscale structure characterized by atomic force microscopy.

Unraveling the multiplex folding of nucleosome chains in higher order chromatin.

The DNA of eukaryotic chromatin and chromosomes is repeatedly supercoiled around histone octamers forming 'beads-on-a-string' chains of nucleosomes. The extent of nucleosome chain folding and DNA accessibility vary between different functional and epigenetic states of nuclear chromatin and change dramatically upon cell differentiation, but the molecular mechanisms that direct 3D folding of the nucleosome chain in vivo are still enigmatic. Recent advances in cell imaging and chromosome capture techniques have radically challenged the established paradigm of regular and hierarchical chromatin fibers by highlighting irregular chromatin organization and the importance of the nuclear skeletal structures hoisting the nucleosome chains. Here, we argue that, by analyzing individual structural elements of the nucleosome chain - nucleosome spacing, linker DNA conformations, internucleosomal interactions, and nucleosome chain flexibility - and integrating these elements in multiplex 3D structural models, we can predict the features of the multiplex chromatin folding assemblies underlying distinct developmental and epigenetic states in living cells. Furthermore, partial disassembly of the nuclear structures suspending chromatin fibers may reveal the intrinsic mechanisms of nucleosome chain folding. These mechanisms and structures are expected to provide molecular cues to modify chromatin structure and functions related to developmental and disease processes.

General Purpose Water Model Can Improve Atomistic Simulations of Intrinsically Disordered Proteins.

Unconstrained atomistic simulations of intrinsically disordered proteins and peptides (IDP) remain a challenge: widely used, "general purpose" water models tend to favor overly compact structures relative to experiment. Here we have performed a total of 93 μs of unrestrained MD simulations to explore, in the context of IDPs, a recently developed "general-purpose" 4-point rigid water model OPC, which describes liquid state of water close to experiment. We demonstrate that OPC, together with a popular AMBER force field ff99SB, offers a noticeable improvement over TIP3P in producing more realistic structural ensembles of three common IDPs benchmarks: 55-residue apo N-terminal zinc-binding domain of HIV-1 integrase ("protein IN"), amyloid β-peptide (Aβ42) (residues 1-42), and 26-reside H4 histone tail. As a negative control, computed folding profile of a regular globular miniprotein (CLN025) in OPC water is in appreciably better agreement with experiment than that obtained in TIP3P, which tends to overstabilize the compact native state relative to the extended conformations. We employed Aβ42 peptide to investigate the possible influence of the solvent box size on simulation outcomes. We advocate a cautious approach for simulations of IDPs: we suggest that the solvent box size should be at least four times the radius of gyration of the random coil corresponding to the IDP. The computed free energy landscape of protein IN in OPC resembles a shallow "tub" - conformations with substantially different degrees of compactness that are within 2 kB T of each other. Conformations with very different secondary structure content coexist within 1 kB T of the global free energy minimum. States with higher free energy tend to have less secondary structure. Computed low helical content of the protein has virtually no correlation with its degree of compactness, which calls into question the possibility of using the helicity as a metric for assessing performance of water models for IDPs, when the helicity is low. Predicted radius of gyration ( R g) of H4 histone tail in OPC water falls in-between that of a typical globular protein and a fully denatured protein of the same size; the predicted R g is consistent with two independent predictions. In contrast, H4 tail in TIP3P water is as compact as the corresponding globular protein. The computed free energy landscape of H4 tail in OPC is relatively flat over a significant range of compactness, which, we argue, is consistent with its biological function as facilitator of internucleosome interactions.

Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays

The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.

The Free Energy Landscape of Internucleosome Interactions and Its Relation to Chromatin Fiber Structure.

The supramolecular chromatin fiber is governed by molecular scale energetics and interactions. Such energetics originate from the fiber’s building block, the nucleosome core particle (NCP). In recent years, the chromatin fiber has been examined through perturbative methods in attempts to extract the energetics of nucleosome association in the fiber. This body of work has led to different results from experiments and simulations concerning the nucleosome–nucleosome energetics. Here, we expand on previous experiments and use coarse-grained simulations to evaluate the energetics inherent to nucleosomes across a variety of parameters in configurational and environmental space. Through this effort, we are able to uncover molecular processes that are critical to understanding the 30 nm chromatin fiber structure. In particular, we describe the NCP–NCP interactions by relying on an anisotropic energetic landscape, rather than a single potential energy value. The attractions in that landscape arise predominantly from the highly anisotropic interactions provided by the NCP histone N-terminal domain (NTD) tails. Our results are found to be in good agreement with recent nucleosome interaction experiments that suggest a maximum interaction energy of 2.69kBT. Furthermore, we examine the influence of crucial epigenetic modifications, such as acetylation of the H4 tail, and how they modify the underlying landscape. Our results for acetylated NCP interactions are also in agreement with experiment. We additionally find an induced chirality in NCP–NCP interactions upon acetylation that reduces interactions which would correspond to a left-handed superhelical chromatin fiber.

Inter-nucleosomal communication between histone modifications for nucleosome phasing.

Combinatorial effects of epigenetic modifications on transcription activity have been proposed as “histone codes”. However, it is unclear whether there also exist inter-nucleosomal communications among epigenetic modifications at single nucleosome level, and if so, what functional roles they play. Meanwhile, how clear nucleosome patterns, such as nucleosome phasing and depletion, are formed at functional regions remains an intriguing enigma. To address these questions, we developed a Bayesian network model for interactions among different histone modifications across neighboring nucleosomes, based on the framework of dynamic Bayesian network (DBN). From this model, we found that robust inter-nucleosomal interactions exist around transcription start site (TSS), transcription termination sites (TTS) or around CTCF binding sites; and these inter-nucleosomal interactions are often involved in transcription regulation. In addition to these general principles, DBN also uncovered a novel specific epigenetic interaction between H2A.Z and H4K20me1 on neighboring nucleosomes, involved in nucleosome free region (NFR) and nucleosome phasing establishment or maintenance. The level of negative correlation between neighboring H2A.Z and H4K20me1 strongly correlate with the size of NFR and the strength of nucleosome phasing around TSS. Our study revealed inter-nucleosomal communications as important players in signal propagation, chromatin remodeling and transcription regulation.

Plug-and-Play Approach for Preparing Chromatin Containing Site-Specific DNA Modifications: The Influence of Chromatin Structure on Base Excision Repair.

The genomic DNA of eukaryotic cells exists in the form of chromatin, the structure of which controls the biochemical accessibility of the underlying DNA to effector proteins. In order to gain an in depth molecular understanding of how chromatin structure regulates DNA repair, detailed in vitro biochemical and biophysical studies are required. However, because of challenges associated with reconstituting nucleosome arrays containing site-specifically positioned DNA modifications, such studies have been limited to the use of mono- and dinucleosomes as model in vitro substrates, which are incapable of folding into native chromatin structures. To address this issue, we developed a straightforward and general approach for assembling chemically defined oligonucleosome arrays (i.e., designer chromatin) containing site-specifically modified DNA. Our method takes advantage of nicking endonucleases to excise short fragments of unmodified DNA, which are subsequently replaced with synthetic oligonucleotides containing the desired modification. Using this approach, we prepared several oligonucleosome substrates containing precisely positioned 2'-deoxyuridine (dU) residues and examined the efficiency of base excision repair (BER) within several distinct chromatin architectures. We show that, depending on the translational position of the lesion, the combined catalytic activities of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) can be either inhibited by as much as 20-fold or accelerated by more than 5-fold within compact chromatin (i.e., the 30 nm fiber) relative to naked DNA. Moreover, we demonstrate that digestion of dU by UDG/APE1 proceeds much more rapidly in mononucleosomes than in compacted nucleosome arrays, thereby providing the first direct evidence that internucleosome interactions play an important role in regulating BER within higher-order chromatin structures. Overall, this work highlights the value of performing detailed biochemical studies on precisely modified chromatin substrates in vitro and provides a robust platform for investigating DNA modifications in chromatin biology.

H4 Tails Potentially Produce the Diversity in the Orientation of Two Nucleosomes

Histone tails play an important role in internucleosomal interaction and chromatin compaction. To understand how the H4 tails are involved in the internucleosomal interaction, an adaptively biased molecular dynamics simulation of 63 models of two stacked nucleosomes, each with the H4 tails in different locations, was carried out. This simulation generated a variety of orientations of the separated nucleosomes depending on the formation of the H4 tail bridge between the H4 tails and the DNA of the neighboring nucleosomes. For the models that showed distinctive orientations of the two nucleosomes, the free energies of the separation of the nucleosomes were further investigated using umbrella sampling simulations. The attractive force between the nucleosomes was estimated from the free energies; the force when two H4 tail bridges formed varied from 36 to 63 pN, depending on the formation of the H4 tail-bridge and the interfacial interaction, whereas the force reduced to 15–18 pN after either one of the H4 tail bridges had broken, regardless of the conformation of the H4 tail. Additional simulations of the nucleosomes show that when the H4 tail was truncated, the force between the nucleosomes became repulsive (from−3 to −7 pN). We concluded that the H4 tails potentially produce the diversity in the orientation of the two nucleosomes, which would contribute to the polymorphism of the chromatin structure.

The tale of a tail: histone H4 acetylation and the repair of DNA breaks

The ability of cells to detect and repair DNA double-strand breaks (DSBs) within the complex architecture of the genome requires co-ordination between the DNA repair machinery and chromatin remodelling complexes. This co-ordination is essential to process damaged chromatin and create open chromatin structures which are required for repair. Initially, there is a PARP-dependent recruitment of repressors, including HP1 and several H3K9 methyltransferases, and exchange of histone H2A.Z by the NuA4-Tip60 complex. This creates repressive chromatin at the DSB in which the tail of histone H4 is bound to the acidic patch on the nucleosome surface. These repressor complexes are then removed, allowing rapid acetylation of the H4 tail by Tip60. H4 acetylation blocks interaction between the H4 tail and the acidic patch on adjacent nucleosomes, decreasing inter-nucleosomal interactions and creating open chromatin. Further, the H4 tail is now free to recruit proteins such as 53BP1 to DSBs, a process modulated by H4 acetylation, and provides binding sites for bromodomain proteins, including ZMYND8 and BRD4, which are important for DSB repair. Here, we will discuss how the H4 tail functions as a dynamic hub that can be programmed through acetylation to alter chromatin packing and recruit repair proteins to the break site.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Synthetic Chromatin Acylation by an Artificial Catalyst System

Summary Histone acetylation is physiologically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) and constitutes a fundamental regulatory element in gene expression. New types of lysine acylation on histones have recently been identified, but it remains unclear how chromatin function is regulated by divergent types of histone acylation and various enzymes. Here, we report on an approach to modulating histone acylation states synthetically without relying on enzymes. We have developed an artificial catalyst system composed of nucleosome-binding catalysts and acyl donors, which preferentially acetylated or malonylated lysines on histone tails and suppressed intra- and inter-nucleosome interactions similarly to HATs. We demonstrate the utility of our approach by identifying a site-selectivity difference between two HDAC isoforms, Sirt1 and Sirt6, and comparing the functions of histone malonylation and acetylation. Our system is applicable to endogenous chromatin without genetic manipulation; thus, it can be used to dissect the complex regulation of chromatin.

Synthetic Posttranslational Modifications: Chemical Catalyst-Driven Regioselective Histone Acylation of Native Chromatin.

Posttranslational modifications (PTMs) of histones play an important role in the complex regulatory mechanisms governing gene transcription, and their dysregulation can cause diseases such as cancer. The lack of methods for site-selectively modifying native chromatin, however, limits our understanding of the functional roles of a specific histone PTM, not as a single mark, but in the intertwined PTM network. Here, we report a synthetic catalyst DMAP-SH (DSH), which activates chemically stable thioesters (including acetyl-CoA) under physiological conditions and transfers various acyl groups to the proximate amino groups. Our data suggest that DSH, conjugated with a nucleosome ligand, such as pyrrole-imidazole-polyamide and LANA (latency-associated nuclear antigen)-peptide, promotes both natural (including acetylation, butyrylation, malonylation, and ubiquitination) and non-natural (azido- and phosphoryl labeling) PTMs on histones in recombinant nucleosomes and/or in native chromatin, at lysine residues close to the DSH moiety. To investigate the validity of our method, we used LANA-DSH to promote histone H2B lysine-120 (K120) acylation, the function of which is largely unknown. H2BK120 acetylation and malonylation modulated higher-order chromatin structures by reducing internucleosomal interactions, and this modulation was further enhanced by histone tail acetylation. This approach, therefore, may have versatile applications for dissecting the regulatory mechanisms underlying chromatin function.

How to Open a Nucleosome

DNA accessibility in the cell nucleus depends on its packaging. The stability of the basic compaction unit, the nucleosome, is regulated through the interactions between DNA and histones. Natural sequence variation along the DNA strongly modulates nucleosome stability. Histone variations, mutations and posttranslational modifications alter intra- and internucleosomal interactions. We are investigating interactions which hold nucleosomes together by measuring distances in reconstituted samples using single molecule Forster energy transfer.

Effects of H2A Histone Variants on DNA Sequence and Nucleosome Structure using Coarse Grain Simulations

Coarse grain molecular dynamics (CG-MD) simulations are seeing a rising tendency in a wide range of applications because their potential to enhance the sampling of all-atomistic simulations (AA-MD) and the capacity to make near-atomistic millisecond-timescale simulations practical, putting the second threshold on the horizon. In this field, the coarse-grained Martini force field has a prominent position and has recently extended its already range of applications to DNA-containing biomolecules [1].Eukaryotic genomic DNA exists as highly compacted nucleosome arrays called chromatin. Each nucleosome contains a 147-base-pair (bp) stretch of DNA, which is sharply bent and tightly wrapped around a histone protein octamer [2]. Several mechanisms regulate DNA accessibility, including replacement of canonical histones with specialized histone variants. Among the core histone variants, the H2A family is the biggest one -to date 5 histones variants are documented- showing also the highest sequence divergence among histone families. H2A variants show striking differences in three sites that are critical in intra- and inter-nucleosome interactions: the docking domain, which is close to the DNA entry/exit region, the L1 loop and the acidic patch, which is involved in nucleosome-nucleosome interactions. We have studied the structural and dynamic differences in those regions among H2A histone variants-containing nucleosome structures.Furthermore, it has been shown that DNA sequence effects vary between nucleosomes resulting in a significant factor in their stability. For this reason, we have also analyzed the differences in the associated DNA flexibility along the wrapped DNA sequence derived from MD simulations.

Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation.

Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains.

Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)+ RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

Chromatin Unfolding by Epigenetic Modifications Explained by Dramatic Impairment of Internucleosome Interactions: A Multiscale Computational Study.

Histone tails and their epigenetic modifications play crucial roles in gene expression regulation by altering the architecture of chromatin. However, the structural mechanisms by which histone tails influence the interconversion between active and inactive chromatin remain unknown. Given the technical challenges in obtaining detailed experimental characterizations of the structure of chromatin, multiscale computations offer a promising alternative to model the effect of histone tails on chromatin folding. Here we combine multimicrosecond atomistic molecular dynamics simulations of dinucleosomes and histone tails in explicit solvent and ions, performed with three different state-of-the-art force fields and validated by experimental NMR measurements, with coarse-grained Monte Carlo simulations of 24-nucleosome arrays to describe the conformational landscape of histone tails, their roles in chromatin compaction, and the impact of lysine acetylation, a widespread epigenetic change, on both. We find that while the wild-type tails are highly flexible and disordered, the dramatic increase of secondary-structure order by lysine acetylation unfolds chromatin by decreasing tail availability for crucial fiber-compacting internucleosome interactions. This molecular level description of the effect of histone tails and their charge modifications on chromatin folding explains the sequence sensitivity and underscores the delicate connection between local and global structural and functional effects. Our approach also opens new avenues for multiscale processes of biomolecular complexes.

Cooperative stabilization of the SIR complex provides robust epigenetic memory in a model of SIR silencing in Saccharomyces cerevisiae

How alternative chromatin-based regulatory states can be made stable and heritable in order to provide robust epigenetic memory is poorly understood. Here, we develop a stochastic model of the silencing system in Saccharomyces cerevisiae that incorporates cooperative binding of the repressive SIR complex and antisilencing histone modifications, in addition to positive feedback in Sir2 recruitment. The model was able to reproduce key features of SIR regulation of an HM locus, including heritable bistability, dependence on the silencer elements, and sensitivity to SIR dosage. We found that antisilencing methylation of H3K79 by Dot1 was not needed to generate these features, but acted to reduce spreading of SIR binding, consistent with its proposed role in containment of silencing. In contrast, cooperative inter-nucleosome interactions mediated by the SIR complex were critical for concentrating SIR binding around the silencers in the absence of barriers, and for providing bistability in SIR binding. SIR-SIR interactions magnify the cooperativity in the Sir2-histone deacetylation positive feedback reaction and complete a double-negative feedback circuit involving antisilencing modifications. Thus, our modeling underscores the potential importance of cooperative interactions between nucleosome-bound complexes both in the SIR system and in other chromatin-based complexes in epigenetic regulation.

Nucleosome positioning and composition modulate in silico chromatin flexibility

The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes—the familiar assemblies of ∼150 DNA base pairs and eight histone proteins—found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the ‘local’ inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and that care must be taken in the choice of models used to interpret the experimental properties of long chromatin fibers.

Metaphase Chromatin Plates Explain the Structure and Physical Properties of Condensed Chromosomes

Previous studies showed that during mitosis chromatin filaments are folded into multilayer plates (1). These structures can be self-assembled from chromatin fragments obtained by micrococcal nuclease digestion of metaphase chromosomes (2). Chromosomes of different animal and plant species show great differences in size (which are dependent on the amount of DNA that they contain), but in all cases chromosomes are elongated cylinders that have relatively similar shape proportions (the length to diameter ratio is approximately 13). It is possible to explain this morphology by considering that chromosomes are self-organizing supramolecular structures formed by stacked layers of planar chromatin having different nucleosome-nucleosome interaction energies in different regions (3). The nucleosomes in the periphery of the chromosome are less stabilized by the attractive interactions with other nucleosomes and this generates a surface potential that destabilizes the structure. Chromosomes are smooth cylinders because this morphology has a lower surface energy than structures having irregular surfaces. The symmetry breaking produced by the different values of the surface energies in the telomeres and in the lateral surface explains the elongated structure of the chromosomes. The results obtained by other authors in nanomechanical studies of chromatin and chromosome stretching have been used to test the proposed supramolecular structure. It is demonstrated quantitatively that internucleosome interactions between chromatin layers can justify the work required for elastic chromosome stretching. Chromosomes can be considered as hydrogels with a lamellar liquid crystal organization. The good mechanical properties of this structure may be useful for the maintenance of chromosome integrity during mitosis. Furthermore this chromatin organization avoids random entanglement of the extremely long genomic DNA molecules in chromosomes.(1) Daban (2011) Micron 42:733-750.(2) Milla and Daban (2012) Biophys J 103:567-575.(3) Daban (2014) J. R. Soc. Interface 11:20131043.

Sumoylated Human Histone H4 Prevents Chromatin Compaction by Inhibiting Long-range Internucleosomal Interactions

The structure of eukaryotic chromatin directly influences gene function, and is regulated by chemical modifications of the core histone proteins. Modification of the human histone H4 N-terminal tail region by the small ubiquitin-like modifier protein, SUMO-3, is associated with transcription repression. However, the direct effect of sumoylation on chromatin structure and function remains unknown. Therefore, we employed a disulfide-directed strategy to generate H4 homogenously and site-specifically sumoylated at Lys-12 (suH4ss). Chromatin compaction and oligomerization assays with nucleosomal arrays containing suH4ss established that SUMO-3 inhibits array folding and higher order oligomerization, which underlie chromatin fiber formation. Moreover, the effect of sumoylation differed from that of acetylation, and could be recapitulated with the structurally similar protein ubiquitin. Mechanistic studies at the level of single nucleosomes revealed that, unlike acetylation, the effect of SUMO-3 arises from the attenuation of long-range internucleosomal interactions more than from the destabilization of a compacted dinucleosome state. Altogether, our results present the first insight on the direct structural effects of histone H4 sumoylation and reveal a novel mechanism by which SUMO-3 inhibits chromatin compaction.