Higher order structure in a short repeat length chromatin

Polynucleosomes from calf brain cortical neurone nuclei have an average repeat length of less than 168 base pairs. The ability of this material to adopt higher order structure has been assessed by various physical techniques. Although containing on average less DNA per nucleosome than is required to form a chromatosome, this short repeat length chromatin folded in an H1 dependent manner to a structure with properties similar to those observed for longer repeat length chromatins such as that of chicken erythrocyte (McGhee, J.D., D.C. Rau, E. Charney, and G. Felsenfeld, 1980, Cell, 22:87-96). These observations are discussed in the context of H1 location in the higher order chromatin fiber.     

Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer length

We have used electric dichroism to study the arrangement of nucleosomes in 30 nm chromatin solenoidal fibers prepared from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies from approximately 10 to approximately 80 bp. Field-free relaxation times are consistent only with structures containing 6 +/- 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, our dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examined. This orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle between 20 degrees-33 degrees to the solenoid axis, thus appears to be a general structural feature of the higher order chromatin fiber.     

A higher order chromatin structure that is lost during differentiation of mouse neuroblastoma cells

Application of differential scanning calorimetry to nuclei from rapidly growing mouse neuroblastoma cells showed a melting profile with four major thermal transitions: I (60 degrees C), II (76 degrees C), III (88 degrees C), and IV (105 degrees C). When neuroblastoma cells were induced to differentiate by serum withdrawal or treatment with sodium butyrate, transition IV disappeared, while transition III increased in magnitude. Comparison was made to nuclei from several types of nondividing cells as well as a number of samples from mature tissues. In rapidly dividing cells the predominant endotherm was IV (105 degrees C), while in nondividing cells, transition III (88 degrees C) predominated the calorimetric profile. Cellular differentiation thus appeared to be accompanied by a major change in chromatin structure, as evidenced by a shift in melting temperature from 105 to 90 degrees C, and this may serve to distinguish the Go phase of the cell cycle from G1.     

Involvement of the globular domain of histone H1 in the higher order structures of chromatin

We have attacked H1-containing soluble chromatin by α-chymotrypsin under conditions where chromatin adopts different structures.Soluble rat liver chromatin fragments depleted of non-histone components were digested with α-chymotrypsin in NaCl concentrations between 0 mm and 500 mm. at pH 7, or at pH 10, or at pH 7 in the presence of 4 m-urea. α-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by α-chymotrypsin can be easily monitored.The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: (1) under conditions of H1 dissociation at 400 mm and 500 mm-NaCl (pH 7 and 10); (2) at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; (3) at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; (4) in the presence of 4 m-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mm and 300 mm-NaCl. Under these conditions H1 is degraded by α-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin.Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for α-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers. Based on the present and earlier observations we propose a model for H1 in which the globular domains eventually together with the N-terminal tails form a backbone in the fiber axis, and the nucleosomes are mainly attached to this polymer by the C-terminal tails.     

Nuclear envelope-limited chromatin sheets (ELCS) and heterochromatin higher order structure

The interphase nucleus and nuclear envelope can acquire a myriad of shapes in normal or pathological cell states. There exist a wide variety of indentations and invaginations, of protrusions and evaginations. It has been difficult to classify and name all of these nuclear shapes and, consequently, a barrier to understanding the biochemical and biophysical causes. This review focuses upon one type of nuclear envelope shape change, named “nuclear envelope-limited chromatin sheets” (ELCS), which appears to involve exaggerated nuclear envelope growth, carrying with it one or more layers of ∼30 nm diameter heterochromatin. A hypothesis on the formation of ELCS is proposed, relating higher order heterochromatin structure in an interphase nucleus, nuclear envelope growth, and nuclear envelope-heterochromatin interactions.     

Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.     

Unravelled nucleosomes,nucleosome beads and higher order structures of chromatin: Influence of non-histone components and histone H1

Effects of non-histone components and histone H1 on the morphology of nucleosomes and chromatin were studied by electron microscopy. Soluble rat liver ehromatin was depleted of non-histone components [NH]or non-histone components and H1 [NH and H1] by dissociation and subsequent fractionation in sucrose gradients in the presence of 300 to 350 mm or 500 mm-NaCl, respectively. In reconstitution experiments the depleted samples were mixed either with [NH] or with [NH and H1] or with purified H1. The morphology of the ionic strength-dependent condensation of the samples was monitored by electron microscopy using 0 mm to 100 mm-NaCl. Based on the appearance of the different types of fibres in very low salt (0 mm up to 10 mm-NaCl), namely the zigzag-shaped, the beads-on-a-string or the DNA-like filaments, it is possible to distinguish between nucleosomes, partially unravelled nucleosomes and unravelled nucleosomes, respectively. Only those fibres which were zigzag-shaped at low ionic strength condense at increasing ionic strength into higher order structures of compact fibres. We demonstrate the dependence of the appearance of nucleosomes and chromatin upon its composition and upon the ionic strength of the solvent.[NH] have no detectable influence upon the formation of higher order chromatin structures, but they can prevent the unravelling of nucleosomes at very low ionic strength, presumably by charge shielding.For the appearance of zigzag-shaped fibres and for the condensation into compact fibres with increasing ionic strength, H1 must be present in about native amounts. Partial removal of H1 (about 10%) promotes a change from fibres into tangles. This supports the model that an H1 polymer is stabilizing the higher order chromatin structures.Reconstitution experiments with purified H1 regenerated fibres containing all the features of [NH]-depleted chromatin. Reconstitution experiments with [NH and H1] promoted fibres compatible with control chromatin. Overloading of chromatin with H1 led to additional condensation. The detailed morphology of the reconstituted fibres showed local distortions. One possibility explaining these local distortions would be competition between “main” and “additional” binding sites for histone H1.     

Comparison of the primary structure of reconstructed minimal nucleosomes and nucleosomes isolated from the chromatin

We have reconstructed nucleosomes from a histone octamer (H2A, H2B, H3, H4)2 and DNA 146 b.p. or 2-3 thousands b.p. in length. Comparison by means of DNA-histone cross-links of the primary organization of minimal nucleosomes obtained by reconstruction or isolated from chromatin of chicken erythrocyte nuclei has demonstrated a high similarity in histone location on their DNAs. Simultaneously, there have been observed some variations in the character of interaction for all core histones with DNA on nucleosomes. Thus, the cross-link of histone H4 with DNA of a core particle at H4 sites (65), unlike H4(55) and H4(88) sites, significantly depends on the superstructure of chromatin, ionic strength of solution and the presence of denaturating agents. All these differences are expected to probe the existence of conformational isomers for core particles. (Bracketed is the distance from the histone interaction site with the DNA of the core particle to the DNA 5'-terminus.)     

The higher order structure of the centromere

The architecture of the centromere region of mouse chromosomes has been studied in cells grown in the presence of 5-azacytidine. This drug interferes with normal condensation producing elongated centromere regions. It has been found that this effect is reversible in the presence of the drug, allowing the observation of the repackaging of the extended centromere into a structure exhibiting native centromere morphology. Light microscopy as well as transmission and scanning electron microscopy of this condensation process suggests that the native centromere is formed by the helical folding of a subfiber with an approximate diameter of 100 nm. This fiber is in turn composed of loops of the 30-nm fiber class. The boundary between successive gyres of the subfiber are obscured at the completion of condensation resulting in the formation of a homogenous 250- to 300-nm fiber that is the native centromere. These observations provide evidence for an additional level of chromatin organization within the metaphase chromosome.     

Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.     

The DNase I sensitive state of "active" globin gene chromatin resists trypsin treatments which disrupt chromatin higher order structure

Active genes in higher eukaryotes reside in chromosomal domains which are more sensitive to digestion by DNase I than the surrounding inactive chromatin. Although it is widely assumed that some modification of higher order structure is important to the preferential DNase I sensitivity of active chromatin, this has so far not been tested. Here we show that the structural distinction between DNase I sensitive and resistant chromatin is remarkably stable to digestion by trypsin. Chick embryonic red blood cell nuclei were subjected to increasing levels of trypsin digestion and then assayed in the following three ways: (1) by gel electrophoresis for histone cleavage, (2) by sedimentation and nuclease digestion for loss of higher order structure, and (3) by dot-blot hybridization to globin and ovalbumin probes for disappearance of preferential DNase I sensitivity. We have found that chromatin higher order structure is lost concomitantly with the cleavage of histones H1, H5, and H3. In contrast, the preferential sensitivity of the globin domain to DNase I persists until much higher concentrations of trypsin, and indeed is not completely abolished even by the highest levels of trypsin we have used. We therefore conclude that the structural distinction of active chromatin, recognized by DNase I, does not reside at the level of higher order structure.     

A repeating unit of higher order chromatin structure in chick red blood cell nuclei

The organization of nucleosomes in higher order chromatin structures has been studied by electron microscopy of chick red blood cell nuclei. Chromatin appears as a thick fiber with an average diameter of approximately 300 Å when prepared for electron microscopy in buffers which approximate physiological ionic strength. Progressive steps of disassembly of the thick fiber into individual nucleosomes could be induced either by ionic strength reduction or by tRNA treatment (which removes histone H1 and some non-histone chromosomal proteins). When disassembly was induced by ionic strength reduction in the presence of Mg⁺⁺ (or Ca⁺⁺), the lengths of the intermediate disassembly products were found to be multiples of 330 Å. The diameter of these structures was estimated to be 275 Å. This intermediate in the disassembly process is not observed if thick fiber disassembly is induced by ionic strength reduction in the absence of divalent cations. To investigate whether the higher order structural unit is present in the thick fiber at physiological ionic strengths, tRNA treatment was used to induce thick fiber disassembly under physiological monovalent ionic conditions. In this case, either with or without divalent cations, a supranucleosomal unit was found with dimensions similar to those given above. This data provides evidence for a slightly oblong supranucleosomal structure (330 × 275 Å) which forms a repeating unit in the chromatin thick fiber.     

Torsional behavior of chromatin is modulated by rotational phasing of nucleosomes

Torsionally stressed DNA plays a critical role in genome organization and regulation. While the effects of torsional stresses on naked DNA have been well studied, little is known about how these stresses propagate within chromatin and affect its organization. Here we investigate the torsional behavior of nucleosome arrays by means of Brownian dynamics simulations of a coarse-grained model of chromatin. Our simulations reveal a strong dependence of the torsional response on the rotational phase angle Ψ₀ between adjacent nucleosomes. Extreme values of Ψ₀ lead to asymmetric, bell-shaped extension-rotation profiles with sharp maxima shifted toward positive or negative rotations, depending on the sign of Ψ₀, and to fast, irregular propagation of DNA twist. In contrast, moderate Ψ₀ yield more symmetric profiles with broad maxima and slow, uniform propagation of twist. The observed behavior is shown to arise from an interplay between nucleosomal transitions into states with crossed and open linker DNAs and global supercoiling of arrays into left- and right-handed coils, where Ψ₀ serves to modulate the energy landscape of nucleosomal states. Our results also explain the torsional resilience of chromatin, reconcile differences between experimentally measured extension-rotation profiles, and suggest a role of torsional stresses in regulating chromatin assembly and organization.     

Non-random arrangement of nucleosomes in satellite I containing chromatin of rat liver.

The location of nucleosomes on the nucleotide sequence of rat satellite I DNA was investigated using micrococcal nuclease, exonuclease III, and restriction nucleases as tools. Hae III cleaved the satellite DNA containing chromatin very preferentially in the linker region. Nucleosomes were found predominantly in three defined positions on the 370 bp satellite I monomer unit. This type of arrangement occurs on not more than half of the satellite DNA containing chromatin while the rest of this chromatin is arranged differently. The arrangement of nucleosomes with high probability in preferred frames and with low probability in less preferred frames may be a general phenomenon which can be discussed as a possible mechanism to modulate sequence recognition.     

Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.