30-nm Fiber Research Articles

Open and closed domains in the mouse genome are configured as 10‐nm chromatin fibres

The mammalian genome is compacted to fit within the confines of the cell nucleus. DNA is wrapped around nucleosomes, forming the classic "beads-on-a-string" 10-nm chromatin fibre. Ten-nanometre chromatin fibres are thought to condense into 30-nm fibres. This structural reorganization is widely assumed to correspond to transitions between active and repressed chromatin, thereby representing a chief regulatory event. Here, by combining electron spectroscopic imaging with tomography, three-dimensional images are generated, revealing that both open and closed chromatin domains in mouse somatic cells comprise 10-nm fibres. These findings indicate that the 30-nm chromatin model does not reflect the true regulatory structure in vivo.

Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding

In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.

Human mitotic chromosome structure: what happened to the 30-nm fibre?

EMBO J 31 7, 1644–1653 February172012 The long-standing view of chromosome packaging is that 10-nm beads-on-a string chromatin fibres fold into higher-order 30-nm fibres, which further twist and coil to form highly condensed chromosomes. After four decades of intense pursuit of the structure and properties of 30-nm chromatin fibres, work of Nishino et al (2012) in this issue of The EMBO Journal demonstrates that regular 30-nm fibres are absent from human mitotic chromosomes. The emerging view is that chromosome-level condensation can be achieved through packaging of 10-nm fibres in a fractal manner.

Quantitative Analysis of the Chromatin Proteome in Disease Reveals Remodeling Principles and Identifies High Mobility Group Protein B2 as a Regulator of Hypertrophic Growth

A fundamental question in biology is how genome-wide changes in gene expression are enacted in response to a finite stimulus. Recent studies have mapped changes in nucleosome localization, determined the binding preferences for individual transcription factors, and shown that the genome adopts a nonrandom structure in vivo. What remains unclear is how global changes in the proteins bound to DNA alter chromatin structure and gene expression. We have addressed this question in the mouse heart, a system in which global gene expression and massive phenotypic changes occur without cardiac cell division, making the mechanisms of chromatin remodeling centrally important. To determine factors controlling genomic plasticity, we used mass spectrometry to measure chromatin-associated proteins. We have characterized the abundance of 305 chromatin-associated proteins in normal cells and measured changes in 108 proteins that accompany the progression of heart disease. These studies were conducted on a high mass accuracy instrument and confirmed in multiple biological replicates, facilitating statistical analysis and allowing us to interrogate the data bioinformatically for modules of proteins involved in similar processes. Our studies reveal general principles for global shifts in chromatin accessibility: altered linker to core histone ratio; differing abundance of chromatin structural proteins; and reprogrammed histone post-translational modifications. Using small interfering RNA-mediated loss-of-function in isolated cells, we demonstrate that the non-histone chromatin structural protein HMGB2 (but not HMGB1) suppresses pathologic cell growth in vivo and controls a gene expression program responsible for hypertrophic cell growth. Our findings reveal the basis for alterations in chromatin structure necessary for genome-wide changes in gene expression. These studies have fundamental implications for understanding how global chromatin remodeling occurs with specificity and accuracy, demonstrating that isoform-specific alterations in chromatin structural proteins can impart these features.

Single Molecule Force Spectroscopy Reveals a Left Handed Helical Folding for the 30 nm Chromatin Fiber

In eukaryotic cells, genetic and epigenetic information is stored in DNA and histone proteins which together form compact chromatin fibers. The structure of chromatin fibers has been heavily debated, but experiments in vitro clearly show folding of arrays of nucleosomes into 30 nm thick fibers. The stability of these fibers and their nucleosomes has been implicated to depend on both force and torque. In previous force spectroscopy experiments, we showed that folding of the fibers depends on the length of the linker DNA between the nucleosomes, and that a 50 bp linker length results in a helical folding. Here we used magnetic tweezers to probe the mechanical response of a single 30 nm fiber under torsion, and found that it is folded in a left handed helix. Applying negative twist stabilizes a fiber against rupture for forces up to 5 pN. Positive twist can compensate the buildup of negative supercoiling when nucleosomes unwrap their DNA at forces above 3 pN and therefore positive twist destabilizes the fiber. These experiments have revealed for the first time the topology of a 30nm chromatin fiber and its response to torque. The results have important implications for the mechanism of DNA based molecular motors that operate in a chromatin context, like the ones involved in chromatin remodeling, transcription and replication.

Biophysical Characterization of a Recombinant Chromatin-Liposome Aggregates

Self-assembled aggregates of recombinant chromatin and liposomes were characterized by synchrotron small angle x-ray spectroscopy (SAXS) and optical microscopy. Recombinant nucleosome core particles (NCP) and 12-mer nucleosome arrays, where half of the negative DNA charge is neutralized by histone octamers, were used as a chromatin model. Liposomes of different charge and charge density were prepared from the either cationic DOTAP or anionic DOPG lipid molecules mixed with zwitterionic DOPC at different molar percentages.Two distinct phases were identified in aggregates with low and high charge density cationic liposomes. The former appeared as translucent precipitate, probably due to higher water retention, compared to latter appearing compact and opaque, and exhibiting birefringence. Systematic SAXS measurements revealed chromatin-lipid bilayer complexes at 3-10% DOTAP, with lamellar distance around 160 A for NCP, and 200 A for arrays. At 10-100% DOTAP, proteins were displaced from DNA by cationic liposomes forming DNA lamellar phase. Confocal microscopy with triple fluorescent labeling displayed homogeneous distribution of DNA, proteins and lipids in both phases at the resolution scale determined by the diffraction limit.Array adopted compacted 30-nm fiber and NCP formed columnar-hexagonal phase in the aggregates with anionic liposomes at 2-50 [Mg2+] revealed by SAXS. These Mg-induced effects are known to occur in the absence of anionic liposomes. Variations of charge density and lipid concentration were found not to influence the dimensions of NCP columnar phase. Lipid molecules may prevent resolubilization of condensed chromatin by neutralizing excess of Mg ions.Chromatin aggregates with liposomes of different charge may serve as a model of the eukaryotic nucleus where chromatin is surrounded by the nuclear envelope. The current results may demonstrate why cationic lipids destabilizing for chromatin are not prevalent in the nucleus, and how anionic membranes may influence the chromatin aggregation state.

Throwing transcription for a loop: expression of the genome in the 3D nucleus

The functional output of the genome is closely dependent on its organization within the nucleus, which ranges from the 10-nm chromatin fiber to the three-dimensional arrangement of this fiber in the nuclear space. Recent observations suggest that intra- and inter-chromosomal interactions between distant sequences underlie several aspects of transcription regulatory processes. These contacts can bring enhancers close to their target genes or prevent inappropriate interactions between regulatory sequences via insulators. In addition, intra- and inter-chromosomal interactions can bring co-activated or co-repressed genes to the same nuclear location. Recent technological advances have made it possible to map long-range cis and trans interactions at relatively high resolution. This information is being used to develop three-dimensional maps of the arrangement of the genome in the nucleus and to understand causal relationships between nuclear structure and function.

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.

Epigenetic marks in zebrafish sperm: insights into chromatin compaction, maintenance of pluripotency, and the role of the paternal genome after fertilization

Epigenetic marks in zebrafish sperm: insights into chromatin compaction, maintenance of pluripotency, and the role of the paternal genome after fertilization

Nucleosome Interactions and Stability in an Ordered Nucleosome Array Model System

Although it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions as well as the dynamics of nucleosome stability are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail-independent, suggesting that well ordered stretches of four or fewer nucleosomes do not fully compact to the 30-nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the inter-array self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for inter-array interactions and suggesting that in vivo multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.

Living without 30 nm chromatin fibers

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.

Dynamic plasticity of large-scale chromatin structure revealed by self-assembly of engineered chromosome regions

Interphase chromatin compaction well above the 30-nm fiber is well documented, but the structural motifs underlying this level of chromatin folding remain unknown. Taking a reductionist approach, we analyzed in mouse embryonic stem (ES) cells and ES-derived fibroblasts and erythroblasts the folding of 10-160-megabase pair engineered chromosome regions consisting of tandem repeats of bacterial artificial chromosomes (BACs) containing approximately 200 kilobases of mammalian genomic DNA tagged with lac operator (LacO) arrays. Unexpectedly, linear mitotic and interphase chromatid regions formed from noncontiguously folded DNA topologies. Particularly, in ES cells, these model chromosome regions self-organized with distant sequences segregating into functionally distinct, compact domains. Transcriptionally active and histone H3K27me3-modified regions positioned toward the engineered chromosome subterritory exterior, with LacO repeats and the BAC vector backbone localizing within an H3K9me3, HP1-enriched core. Differential compaction of Dhfr and alpha- and beta-globin transgenes was superimposed on dramatic, lineage-specific reorganization of large-scale chromatin folding, demonstrating a surprising plasticity of large-scale chromatin organization.

Nucleosome Interaction Surface of Linker Histone H1c Is Distinct from That of H10

The fully organized structure of the eukaryotic nucleosome remains unsolved, in part due to limited information regarding the binding site of the H1 or linker histone. The central globular domain of H1 is believed to interact with the nucleosome core at or near the dyad and to bind at least two strands of DNA. We utilized site-directed mutagenesis and in vivo photobleaching to identify residues that contribute to the binding of the globular domain of the somatic H1 subtype H1c to the nucleosome. As was previously observed for the H1(0) subtype, the binding residues for H1c are clustered on the surface of one face of the domain. Despite considerable structural conservation between the globular domains of these two subtypes, the locations of the binding sites identified for H1c are distinct from those of H1(0). We suggest that the globular domains of these two linker histone subtypes will bind to the nucleosome with distinct orientations that may contribute to higher order chromatin structure heterogeneity or to differences in dynamic interactions with other DNA or chromatin-binding proteins.

Role of Direct Interactions between the Histone H4 Tail and the H2A Core in Long Range Nucleosome Contacts

In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To determine whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide cross-linking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand association but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Additionally, we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermolecular self-association.

Chemical Approaches for Studying Histone Modifications

Histones form the protein core around which genomic DNA is wrapped in eukaryotic chromatin. Numerous genetic studies have established that the structure and transcriptional state of chromatin are closely related to histone post-translational modifications. Further elucidation of the precise mechanistic roles for individual histone modifications requires the ability to isolate and study homogeneously modified histones. However, the highly heterogeneous nature of histone modifications in vivo poses a significant challenge for such studies. Chemical tools that have enabled biochemical and biophysical studies of site-specifically modified histones are the focus of this minireview.

Changes in Chromatin Fiber Density as a Marker for Pluripotency

Extensive alterations in chromatin structure at the nucleosome level are linked to developmental potential. We hypothesize that such alterations in chromatin structure reflect and, to some extent, depend on the large-scale reorganization of the nuclear landscape. We have used electron spectroscopic imaging (ESI) to visualize chromatin organization at the mesoscale level of resolution in both pluripotent and differentiated cell types. Pluripotent cells are characterized by a highly dispersed mesh of 10-nm chromatin fibers that fill the nuclear volume. In contrast, differentiated cells display a propensity to form compact chromatin domains that lead to large regions of the nucleus devoid of DNA. Surprisingly, ESI combined with tomography methods reveals that the compact chromatin domains consist of 10-nm rather than 30-nm chromatin fibers. We propose that the transition between compact silent chromatin and open transcriptionally poised or active chromatin is based on the modulation of the packing density of 10-nm fibers rather than a transition between 10- and 30-nm fiber types.

Theoretical Modeling of Protein Accessibility to the Chromatin Fiber

Gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of DNA to RNA. Regulatory proteins function by locating specific sequences of DNA and binding to these sequences to form the transcription initiation complex. The eukaryotic genome is tightly packaged into a dense chromatin fiber. This packaged structure acts both to store the massive genome and to facilitate the accessibility of the genome to regulatory proteins. The interplay between the packaging proteins and the regulatory proteins is critical in normal cellular function and plays a pivotal role in a number of human diseases. We present a theoretical study on the dynamic accessibility of the chromatin fiber, providing an overview of several approaches to this multi-faceted problem. Our modeling efforts address both the structural properties of packaged DNA and the dynamic processes involved in target-site localization of regulatory proteins. Modeling of the 30-nm fiber reveals the impact of local nucleosome configurational properties on the fiber geometry, and we predict the mechanical properties of the assemblies and the resulting dynamic accessibility. We also discuss the role of linker histones and variant histones in the fiber assembly. Using our 30-nm fiber models, we address large scale condensation by epigenetic factors and discuss the role of histone methylation in the determination of heterochromatin and euchromatin states. Upon establishing our chromatin model, we study the dynamic processes involved in the target-site search of regulatory proteins within the complex chromatin structure. This effort combines our structural models with our protein-transport models in order to provide a novel perspective on regulatory-protein function.

Insights into Interphase Large-Scale Chromatin Structure from Analysis of Engineered Chromosome Regions

How chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions that preserve large-scale chromatin structure. Our results confirm the existence of large-scale chromatin domains and fibers formed by the folding of 10-nm and 30-nm chromatin fibers into larger, spatially distinct domains. Transcription at levels within severalfold of the levels measured for endogenous loci occur within these large-scale chromatin structures on a condensed template linearly compacted several hundred fold to 1000-fold relative to B-form DNA. However, transcriptional induction is accompanied by a severalfold decondensation of this large-scale chromatin structure that propagates hundreds of kilobases beyond the induced gene. Examination of engineered chromosome regions in mouse embryonic stem cells (ESCs) and differentiated cells suggests a surprising degree of plasticity in this large-scale chromatin structure, allowing long-range DNA interactions within the context of large-scale chromatin fibers. Recapitulation of gene-specific differences in large-scale chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture.

Multimerization and H3K9me3 Binding Are Required for CDYL1b Heterochromatin Association

Proteins containing defined recognition modules mediate readout and translation of histone modifications. These factors are thought to initiate downstream signaling events regulating chromatin structure and function. We identified CDYL1 as an interaction partner of histone H3 trimethylated on lysine 9 (H3K9me3). CDYL1 belongs to a family of chromodomain factors found in vertebrates. We show that three different splicing variants of CDYL1, a, b, and c, are differentially expressed in various tissues with CDYL1b being the most abundant variant. Although all three splicing variants share a common C-terminal enoyl-CoA hydratase-like domain, only CDYL1b contains a functional chromodomain implicated in H3K9me3 binding. A splicing event introducing an N-terminal extension right at the beginning of the chromodomain of CDYL1a inactivates its chromodomain. CDYL1c does not contain a chromodomain at all. Although CDYL1b displays binding affinity to methyl-lysine residues in different sequence context similar to chromodomains in other chromatin factors, we demonstrate that the CDYL1b chromodomain/H3K9me3 interaction is necessary but not sufficient for association of the factor with heterochromatin. Indeed, multimerization of the protein via the enoyl-CoA hydratase-like domain is essential for H3K9me3 chromatin binding in vitro and heterochromatin localization in vivo. In agreement, overexpression of CDYL1c that can multimerize, but does not interact with H3K9me3 can displace CDYL1b from heterochromatin. Our results imply that multimeric binding to H3K9me3 by CDYL1b homomeric complexes is essential for efficient chromatin targeting. We suggest that similar multivalent binding stably anchors other histone modification binding factors on their target chromatin regions.

High resolution imaging of changes in the structure and spatial organization of chromatin, γ-H2A.X and the MRN complex within etoposide-induced DNA repair foci

The focal accumulation of DNA repair factors, including the MRE11/Rad50/NBS1 (MRN) complex and the phospho-histone variant γ-H2A.X, is a key cytological feature of the DNA damage response (DDR). Although these foci have been extensively studied by light microscopy, there is comparatively little known regarding their ultrastructure. Using correlative light microscopy and electron spectroscopic imaging (LM/ESI) we have characterized the ultrastructure of chromatin and DNA repair foci within the nuclei of normal human fibroblasts in response to DNA double-strand breaks (DSBs). The induction of DNA DSBs by etoposide leads to a global decrease in chromatin density, which is accompanied by the formation of invaginations of the nuclear envelope as revealed by live-cell microscopy. Using LM/ESI and the immunogold localization of γ-H2A.X and MRE11 within repair foci, we also observed decondensed 10nm chromatin fibers within repair foci and the accumulation of large non-chromosomal protein complexes over three hours recovery from etoposide. At 18 h after etoposide treatment, we observed a close juxtapositioning of PML nuclear bodies and late repair foci of γ-H2A.X, which exhibited a highly organized chromatin arrangement distinct from earlier repair foci. Finally, the dual immunogold labeling of MRE11 with either γ-H2A.X or NBS1 revealed that γ-H2A.X and the MRN complex are sub-compartmentalized within repair foci at the sub-micron scale. Together these data provide the first ultrastructural comparison of γ-H2A.X and MRN DNA repair foci, which are structurally dynamic over time and strikingly similar in organization.