Chromatin structure. Evidence that the 30-nm fiber is a helical coil with 12 nucleosomes/turn

Sedimentation analysis has been used to compare the structure of 30-nm chromatin fibers, isolated and digested under conditions that maintain the native structure, with relaxed-refolded chromatin. The native chromatin fibers show sharp, ionic strength-dependent changes in sedimentation coefficient that are not apparent in relaxed-refolded fibers. The first transition at approximately 20 mM ionic strength reflects the organization of the 10-nm polynucleosome chain into a loose helically coiled 30-nm fiber. Between 20 and 60 mM ionic strength there is considerable interaction between nucleosomes within the coils to generate a stable helical array with 12 nucleosomes/turn. Above 60 mM ionic strength the helical coil continues to condense until it precipitates at ionic strengths slightly greater than those considered physiological, indicating that there is no end point in fiber formation. The data is incompatible with a solenoid model with 6 nucleosomes/turn and also rules out the existence of a beaded subunit structure.     

Higher order structure of chromatin: evidence from photochemically detected linear dichroism

We have used photochemically detected linear dichroism to measure the separate average angular orientations of nucleosomes and linker DNA in 30-nm chromatin fibers of varying linker size (20-80 base pairs). Our results indicate that the average tilt angles vary with linker size, but not in a monotonic manner, suggesting that the constancy of geometry of the 30-nm fiber is maintained by compensatory changes of nucleosomal tilt which accommodate packing of variable lengths of linker DNA. We discuss the compatibility of our results with the various classes of models that have been proposed for the 30-nm fiber, including the continuous solenoid model and models built from the basic unit of the zig-zag ribbon. Many models can be eliminated, and all have to be modified to fit our results for chromatins with very long linkers.     

Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure

We have used light microscopy and serial thin-section electron microscopy to visualize intermediates of chromosome decondensation during G1 progression in synchronized CHO cells. In early G1, tightly coiled 100-130-nm "chromonema" fibers are visualized within partially decondensed chromatin masses. Progression from early to middle G1 is accompanied by a progressive uncoiling and straightening of these chromonema fibers. Further decondensation in later G1 and early S phase results in predominantly 60-80-nm chromonema fibers that can be traced up to 2-3 microns in length as discrete fibers. Abrupt transitions in diameter from 100-130 to 60-80 nm along individual fibers are suggestive of coiling of the 60-80-nm chromonema fibers to form the thicker 100-130-nm chromonema fiber. Local unfolding of these chromonema fibers, corresponding to DNA regions tens to hundreds of kilobases in length, reveal more loosely folded and extended 30-nm chromatin fibers. Kinks and supercoils appear as prominent features at all observed levels of folding. These results are inconsistent with prevailing models of chromosome structure and, instead, suggest a folded chromonema model of chromosome structure.     

A polymer model for large-scale chromatin organization in lower eukaryotes

A quantitative model of large-scale chromatin organization was applied to nuclei of fission yeast Schizosaccharomyces pombe (meiotic prophase and G2 phase), budding yeast Saccharomyces cerevisiae (young and senescent cells), Drosophila (embryonic cycles 10 and 14, and polytene tissues) and Caenorhabditis elegans (G1 phase). The model is based on the coil-like behavior of chromosomal fibers and the tight packing of discrete chromatin domains in a nucleus. Intrachromosomal domains are formed by chromatin anchoring to nuclear structures (e.g., the nuclear envelope). The observed sizes for confinement of chromatin diffusional motion are similar to the estimated sizes of corresponding domains. The model correctly predicts chromosome configurations (linear, Rabl, loop) and chromosome associations (homologous pairing, centromere and telomere clusters) on the basis of the geometrical constraints imposed by nuclear size and shape. Agreement between the model predictions and literature observations supports the notion that the average linear density of the 30-nm chromatin fiber is approximately 4 nucleosomes per 10 nm contour length.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro

In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.     

Depletion Effects Massively Change Chromatin Properties and Influence Genome Folding

We present a Monte Carlo model for genome folding at the 30-nm scale with focus on linker-histone and nucleosome depletion effects. We find that parameter distributions from experimental data do not lead to one specific chromatin fiber structure, but instead to a distribution of structures in the chromatin phase diagram. Depletion of linker histones and nucleosomes affects, massively, the flexibility and the extension of chromatin fibers. Increasing the amount of nucleosome skips (i.e., nucleosome depletion) can lead either to a collapse or to a swelling of chromatin fibers. These opposing effects are discussed and we show that depletion effects may even contribute to chromatin compaction. Furthermore, we find that predictions from experimental data for the average nucleosome skip rate lie exactly in the regime of maximum chromatin compaction. Finally, we determine the pair distribution function of chromatin. This function reflects the structure of the fiber, and its Fourier-transform can be measured experimentally. Our calculations show that even in the case of fibers with depletion effects, the main dominant peaks (characterizing the structure and the length scales) can still be identified.     

Interphase epichromatin: last refuge for the 30-nm chromatin fiber?

“Interphase epichromatin” describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of “exposed” acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed “unit threads,” frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with ~ 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a “sandwich” around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a “close-to-native” situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

     

Organization of chromatin during spermiogenesis: beaded fibers,partly beaded fibers,and loss of nucleosomal structure

The aggregation of chromatin during spermiogenesis in the house cricket and many other animals is an orderly process involving the formation of a series of long, thick, well defined structures. The differentiation of chromatin preliminary to the development of such unusual structures is given attention here. Examination of nuclei after lysis and spreading indicated that fibers with closely spaced nucleosomes, like the fibers of somatic chromatin, make up the chromatin in all stages of early spermiogenesis and most of middle spermiogenesis. The thick structures of late spermatids cannot be formed by aggregation of fibers of this somatic type, however; just before thick structures form, chromatin fibers lose the nucleosomal structure. During the process, fibers with nucleosomes spaced at irregular intervals and with long stretches of smooth thin fiber are found, as if nucleosomes at one site on a fiber are broken down independently of those at adjacent sites. Since prior studies of cricket proteins have indicated that somatic histones persist during the stages when nucleosome structure disappears, the observations imply that the histones which are organized in nucleosomes during early stages must become incorporated into different kinds of nucleoprotein complexes during succeeding stages of spermiogenesis.     

Gene functioning and storage within a folded genome

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 organization - the 30 nm fiber

Despite over 30 years of work, the fundamental structure of eukaryotic chromatin remains controversial. Here, we review the roots of this controversy in disparities between results derived from studies of chromatin in nuclei, chromatin isolated from nuclei, and chromatin reconstituted from defined components. Thanks to recent advances in imaging, modeling, and other approaches, it is now possible to recognize some unifying principles driving chromatin architecture at the level of the ubiquitous '30 nm' chromatin fiber. These suggest that fiber architecture involves both zigzag and bent linker motifs, and that such heteromorphic structures facilitate the observed high packing ratios. Interactions between neighboring fibers in highly compact chromatin lead to extensive interdigitation of nucleosomes and the inability to resolve individual fibers in compact chromatin in situ.     

The three-dimensional structure of in vitro reconstituted <Emphasis Type="Italic">Xenopus laevis</Emphasis> chromosomes by EM tomography

We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed that the 30–40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on their topology and high degree of interconnectedness, it is unlikely that 30–40-nm domains arise from the folding of local stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments. By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network, while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30–40-nm chromatin domains. These observations suggest that 30–40-nm domains are essential for establishing long-range chromatin associations that are central for chromosome condensation. Electronic supplementary material The online version of this article (doi ) contains supplementary material, which is available to authorized users.     

The layered organization of nucleosomes in 30 nm chromatin fibers

We have used electron microscopy and established methods of three-dimensional reconstruction to obtain structural information on the 30 nm chromatin fibers from sea cucumber sperm and chicken erythrocytes. The fibers show a longitudinal periodicity of 10–11 nm. We have interpreted this periodicity as due to a grouping of nucleosomes into disks, each disk containing about 5–6 nucleosomes. These disks are closely stacked to form the chromatin fiber. We have built a detailed model for four fibers and we have determined the approximate coordinates of all the nucleosomes in them. The average distance found between neighboring nucleosomes has a value close to 11 nm. They may be connected either as a regularly distorted helix or as a layered zigzag. The second model appears more appropriate, since in the constrictions of the fibers the nucleosomes can only be connected as a zigzag.