Nucleosomes along the DNA form a chromatin fiber
Individual nucleosomes are organized into higher-level structures called chroma tin fibers. There is convincing evidence that certain DNA sequences can provide strong, specific sites for nucleosome positioning (Figure 1A). What is perhaps most interesting is where nucleosomes are not found. The ENCODE project has located millions of nuclease-hypersensitive sites; DNase I map ping indicates that these sites are nucleosome-free. Some of these are at transcription start sites and are flanked by two strongly positioned nucleosomes. There is also evidence for what has been called statistical positioning, where one or more such strongly positioned nucleosome(s), often called index nucleosomes, provide boundary elements for the positioning of a series of spaced nucleosomes, with a regularity that decreases upon moving further from the index nucleosome (Figure 1B).
Fig1. Nucleosome positioning. (A) Effect of nucleoside sequences on nucleosome positioning. Graphs show the fraction of AA, TT, and TA dinucleotides at each position of center-aligned chicken and yeast nucleosome-bound genomic sequences, indicating 10-bp periodicity of these dinucleotides in naturally occurring nucleosomes. For example, mononucleosomes were purified from log-phase yeast Saccharomyces cerevisiae cells following nuclease digestion, the DNA was extracted, and protected fragments of length ~147 bp were cloned and sequenced. (B) Typical nucleosomal occupancy map showing clear demarcation of the gene locus, identifiable by the presence of the 5′ and 3′ nucleosome-depleted regions or NDR. The 5′ NDR is flanked by rather tightly bound nucleosomes downstream of the transcription start site. [A, adapted from Segal E, Fondufe-Mittendorf Y, Chen L et al. (2006) Nature 442: 772–778. With permission from Macmillan Publishers, Ltd. B, adapted, courtesy of Sergei Grigoryev, Penn State University, State College, Pennsylvania, USA, and Gaurav Arya, University of California, San Diego, California, USA.]
Despite some such specific positioning, the spacing of nucleosomes on the DNA is generally irregular. As shown by nuclease digestion experiments, the average spacing varies between species and cell types, from about 160 to 240 bp. In most cases, this is considerably larger than the 147 bp carried on the nucleosome core particle, and so there must be linker DNA between particles. This is what pro duces the beads-on-a-string appearance noted by early electron microscopists. This linker DNA turns out to be the habitat of the fifth class of histones, the linker histones (see Table 1). Together with the core particle, they form what is called the chromatosome. Linker histones bind to the linker DNA entering and exiting the nucleosome in a manner that stabilizes the particle (Figure 2). In addition, these histones seem to play important roles in the formation and stabilization of higher-order folding of the chromatin fiber. Such folding is essential to give sufficient compaction of the chromatin in the nucleus. Note that formation of a nucleosome only compacts about 40 nm of DNA into a 10 nm particle. This compaction ratio of 4:1 is far from the values in the thousands necessary for proper compaction in the nucleus.
Table1. Histones: five major classes. The linker histones, also known as H1 histones, are a family of closely related members; they bind to the linker DNA between nucleosomal particles. The core histones derive their names from their structural role as organizers of the core particle. The sizes, amino acid compositions, and sequences of these histones vary somewhat from species to species; numbers in the table refer to bovine histones. Still, histones are among the most highly conserved proteins in evolution.
Fig2. Chromatosome: a particle containing linker histones and more than 147 bp of DNA. (A) Linker histones, LH, bind to the nucleosome at the entry–exit point of nucleosomal DNA. Each member of the linker histone family possesses a short unstructured N-terminus, a globular domain (winged-helix motif), and a long unstructured C-tail. The binding of an isolated globular domain seals off two full turns of DNA around the histone octamer, compared to the 1.67 turn in the particle lacking LH. Interaction of the C-tail with the incoming and outgoing linker DNA creates the stem structure. The EM images above the two schematics are of nucleosomes reconstituted from naked DNA and histone octamers in the absence or presence of LHs. The stem structure has also been seen in situ. (B) To-scale schematic of the chromatosome based on the crystal structure of the core particle, the known dimensions of the globular domain GH5 of linker histones, and the expected trajectory of the two linker DNA segments. [A, EM images from Hamiche A, Schultz P, Ramakrishnan V et al. (1996) J Mol Biol 257: 30–42. With permission from Elsevier. B, adapted from Leuba SH, Bustamante C, van Holde K et al. (1998) Biophys J 74: 2830–2839. With permission from Elsevier.]
The chromatin fiber is folded, but its structure remains controversial
Does the chromatin fiber have a regular structure; and if so, what is it? That question has perplexed researchers for almost half a century. It has proved very difficult to visualize the chromatin fibers as they are packed into the interphase nucleus in any interpretable way. The crowding of macromolecules is just too great. Therefore, from the beginning of modern chromatin research, around 1970, investigators have utilized a variety of methods to extract, or to attempt to reconstruct, what appears to be at least a basic element in that structure. A host of studies using a wide variety of physical techniques, ranging from extraction of chromatin fiber from gently digested lysed nuclei to reconstruction of regularly spaced nucleosome arrays on repeated sequence DNA, have given a superficially similar result: a more or less regular helical fiber of about 30 nm in diameter. This 30-nm fiber has been observed in a number of electron microscopic studies. Some typical views are shown in Figure 3, as are some AFM images taken under similar conditions.
Fig3. Micrographs of native chromatin fibers imaged by (A) electron microsopy, EM or (B) atomic force microscopy, AFM. In both cases, isolated fibers were fixed with glutaraldehyde under various salt concentrations, dialyzed against buffers that do not contain salt, and imaged. Compaction of the fiber with increasing ionic strength is clearly visible. [A, from Thoma F, Koller T & Klug A (1979) J Cell Biol 83: 403–427. With permission from The Rockefeller University Press. B, from Zlatanova J, Leuba SH, Yang G et al. (1994) Proc Natl Acad Sci USA 91: 5277–5280. With permission from National Academy of Sciences, USA.]
We can say a few things about the structure of chromatin fibers with some confidence:
• It probably has physiological significance, since packed structures like this can be occasionally glimpsed in sectioned nuclei.
• It is stabilized by physiological salt concentration and especially by divalent ions.
• It is stabilized by the linker histones.
Beyond these areas of agreement, there is discord, even after decades of research. The main arguments are on a very fundamental point: what kind of a helix is this? The original proposal, by Mellema and Klug in 1976, was for a left-handed solenoidal helix with linker DNA passing directly from one nucleosome to the next along the spiral of the helix. But others have proposed two-start helices, or helices with com plex internal connections. A few of the many models that have been proposed are shown in Figure 4. As one can see from these models, the proposed structures will be difficult to distinguish by simple electron microscopic observation: they look pretty much alike unless observed in fine detail.
Fig4. Models depicting the possible arrangement of nucleosomes in the 30-nm fiber. Note that none of the models has been universally accepted. (A) Side and top views of two major types of models: a one-start helix, or solenoid, and a two-start helix. A two-start helix can be visualized as follows: Imagine a zigzag ribbon of nucleosomes, where linkers pass back and forth between two rows of nucleosome particles. Now twist this ribbon into a helix. Nucleosomes are represented in both structures by cylinders. The linker DNA is not shown in the side view of the solenoid, since in most models it is located in the fiber interior; the linkers can be seen from the outside only in the two-start helical ribbon, where they help to create the zigzag ribbon of two rows of nucleosomes. Different color is used to indicate an example of nucleosomes, designated n and n+1, that are next to each other in the extended chromatin fiber; note that these nucleosomes have different spatial locations, and different neighbors, in the different models for the 30-nm fiber. The red lines are simply to emphasize the helical nature of both structures. (B) Straight-linker model: successive nucleosomes in the fiber are connected by straight linkers that criss-cross the fiber interior. The nucleosomes are situated at the periphery, as in the helical models. Further condensation will yield a lumpy, irregular fiber, like the one in AFM images at 80 mM NaCl. [A, adapted from van Holde K & Zlatanova J (2007) Semin Cell Dev Biol 18: 651–658. With permission from Elsevier. B, adapted, courtesy of Mikhail Karymov, California Institute of Technology, Pasadena, California, USA.]
There is also the fundamental question as to whether any of the models proposed, or indeed any uniform model, has any relevance to chromatin in vivo. Certainly, pieces of 30-nm fiber extracted from nuclei rarely show a smooth, regular helical structure; they are lumpy and bent. This may be what we should expect, as we know that linker lengths are locally heterogeneous, and this fact should argue against any wholly regular structure. Indeed, AFM images of chromatin being progressively compacted do not seem to converge on a single regular structure (see Figure 2B).
The organization of chromosomes in the interphase nucleus is still obscure
Early cytological studies of interphase nuclei revealed the existence of two readily distinguishable forms of chromatin structure: highly condensed heterochromatin and more dispersed euchromatin. These distinctions are still recognized and are thought to correlate with transcriptional activity, with transcription of the condensed heterochromatin being repressed. There also seem to be differences in the overall protein composition of these regions and in the presence of specific histone post-translational modifications. Newer techniques are able to show that the linear double-stranded DNA molecules that constitute individual interphase chromosomes occupy distinct portions of the nuclear volume, forming chromosome territories (Figure 5). The chromatin fiber within each territory can form individual loop domains, and domains that make temporal excursions out of their respective territories can be bridged together, presumably to allow their coordinated regulation through interactions with transcriptional factories. A significant portion of the chromatin fiber is associated with the lamina structure in the so-called lamina-associated domains, which are transcriptionally repressed. Intra- and interchromosomal interactions also form distinct nucleoli that contain both active and inactive copies of tandemly repeated ribosomal genes. A final recognizable structure that may have a role in the spatial and topological organization of the genome is the nuclear matrix, an insoluble meshwork of various skeletal proteins.
Fig5. Chromosome territories in the interphase nucleus. (A) Organization of individual interphase chromosome territories (CTs) denoted here as Chr. A, Chr. B, and Chr. C, and shown in different colors. (B) Light optical section through a chicken fibroblast nucleus, showing mutually exclusive CTs. The individual chromosome territories were painted by a combinatorial scheme of several compounds, which were then detected by secondary antibodies labeled with the fluorescent dyes Cy3, fluorescein isothiocyanate (FITC), and Cy5. The two homologous chromosomes comprising each chromosome pair are seen in separate locations; note that only one of the two territories for each of chromosome 4 and chromosome 6 is displayed in this section. (C) Three-dimensional computational model of chromosome territories, visualized by use of pseudocolors, in a male diploid human cell nucleus. [B, from Cremer T & Cremer C (2001) Nat Rev Genet 2: 292–301. With permission from Macmillan Publishers, Ltd. Image courtesy of Felix A. Habermann. C, from Cremer T, Cremer M, Dietzel S et al. (2006) Curr Opin Cell Biol 18: 307–316. With permission from Elsevier.]
A curious, and still poorly understood, phenomenon is the distribution of gene-rich and gene-poor chromosomes within the nuclear volume. The gene poor chromosomes tend to locate at the nuclear periphery, whereas gene-rich chromosomes prefer the nuclear interior (Figure 6).
Fig6. Gene-rich and gene-poor chromosome territories in human cells. (A) Lymphocyte nucleus: three-dimensional recon structions of gene-poor chromosome 18 territory, shown in red, and gene-rich chromosome 19 territory, shown in green. Chromosome 18 territories were typically found at the nuclear periphery, whereas chromosome 19 territories were located in the nuclear interior. Left, an X, Y view: a section of the nucleus is shown in gray. Only the parts of the territories below this section can be seen. Right, an X, Z view: the arrow marks the side from which the section is viewed. (B) Three-dimensional reconstructions of the same chromosomes in three different types of cells. Part of the nuclear border is shown: outside, blue; inside, silver-gray. Usually, CT19 is situated in the nuclear interior, whereas CT18 is found at the nuclear periphery, either side-by-side or at remote sites. Frequently, this distribution is lost in tumor cells. [A, from Cremer T & Cremer C (2001) Nat Rev Genet 2: 292–301. With permission from Macmillan Publishers, Ltd. Image courtesy of Irina Solovei, Ludwig-Maximilians-Universität, Munich, Germany. B, from Cremer M, Küpper K, Wagler B et al. (2003) J Cell Biol 162: 809–820. With permission from The Rockefeller University Press.]
