Octamers, histone

There are five separate histone isoforms histones HI, H2A, H2B, H3, and H4. Histones H2A, H2B, H3, and H4 assemble as dimers to form an octamer (2 x 2A -i- 2 x 2B -i- 3 -i- 4 = 8). The DNA wraps 1.75 turns around this octamer. Histone HI acts as a between the octameric units. When viewed ruider the electron microscope, the packaging yields a stracture that is reminiscent of a string of beads in which octamers and associated DNA are the beads and the linker is the HI boruid to DNA. A bead and its linker are referred to as a nucleosome. 

Histone core octamer (here shown in cross section)... 

FIGURE 11.23 A diagram of the histone octamer. Nucleosomes consist of two turns of DNA supercoiled about a histone core octamer. 

If chromatin is swelled suddenly in water and prepared for viewing in the electron microscope, the nucleosomes are evident as beads on a string, dsDNA being the string (Figure 12.28). The structure of the histone octamer core has been determined by X-ray crystallography without DNA by E. N. Moudrianakis s laboratory (Figure 12.29) and wrapped with DNA by T. J. 

Richmond and collaborators (Figure 12.30). The octamer (Figure 12.29) has surface landmarks that guide the course of the DNA around the octamer 146 bp of B-DNA in a flat, left-handed superhelical conformation make 1.65 turns around the histone core (Figure 12.30), which itself is a protein superhelix consisting of a spiral array of the four histone dimers. Histone 1, a three-domain protein, serves to seal the ends of the DNA turns to the nucleosome core and to organize the additional 40 to 60 bp of DNA that link consecutive nucleo-... 

FIGURE 12.31 A model for chromosome structure, human chromosome 4. The 2-um DNA helix is wound twice around histone octamers to form 10-um uucleosomes, each of which contains 160 bp (80 per turn). These uucleosomes are then wound in solenoid fashion with six uucleosomes per turn to form a 30-nm filament. In this model, the 30-nm filament forms long DNA loops, each containing about 60,000 bp, which are attached at their base to the nuclear matrix. Eighteen of these loops are then wound radially around the circumference of a single turn to form a miniband unit of a chromosome. Approximately 10 of these minibands occur in each chromatid of human chromosome 4 at mitosis. 

Chromatin is composed of nucleosomes, where each comprise 147 base pairs of DNA wrapped around an octamer oftwo copies of each histone H2A, H2B, H3, and H4. Nucleosomes are folded into higher-order structures that are stabilized by linker histones. Chromatin structure can be altered by enzymes that posttranslationally modify histones (e.g., through phosphorylation, acetylation, methylation, or ubiquitination) or by ATP-driven chromatin-remodeling complexes that alter nucleosome position and/or composition. 

Histones are small, basic proteins required to condense DNA into chromatin. They have been first described and named in 1884 by Albrecht Kossel. There are five main histones HI, H2A, H2B, H3 andH4. An octamer of core histones H2A, H2B, H3 andH4 is located inside a nucleosome, the central building block of chromatin, with about 150 base pairs of DNA wrapped around. The basic nature of histones, mediated by the high content of lysine and arginine residues, allows a direct interaction with the acidic phosphate back bone of DNA. The fifth histone HI is located outside at the junction between nucleosomes and is referred to as the linker histone. Besides the main histones, so-called histone variants are known, which replace core histones in certain locations like centromers. 

The nucleosome represents the first level of DNA condensation and is the basic building block of all chromatin structures. It was discovered in 1973 and consists of a central histone octamer with about 150 base pairs of DNA wrapped around. 

The core unit of the chromatin, the nucleosome, consists of histones arranged as an octamer consisting of a (H3/ H4)2-tetramer complexed with two histone H2A/H2B dimers. Accessibility to DNA-binding proteins (for replication, repair, or transcription) is achieved by posttranslational modifications of the amino-termini of the histones, the histone tails phosphorylation, acetylation, methylation, ubiquitination, and sumoyla-tion. Especially acetylation of histone tails has been linked to transcriptional activation, leading to weakened interaction of the core complexes with DNA and subsequently to decondensation of chromatin. In contrast, deacetylation leads to transcriptional repression. As mentioned above, transcriptional coactivators either possess HAT activity or recruit HATs. HDACs in turn act as corepressors. 

When the histone octamer is mixed with purified, double-stranded DNA, the same x-ray diffraction pattern is formed as that observed in freshly isolated chromatin. Electron microscopic studies confirm the existence of reconstituted nucleosomes. Furthermore, the reconsti-mtion of nucleosomes from DNA and histones H2A, H2B, H3, and H4 is independent of the organismal or cellular origin of the various components. The histone HI and the nonhistone proteins are not necessary for the reconstitution of the nucleosome core. 

In the nucleosome, the DNA is supercoiled in a left-handed helix over the surface of the disk-shaped histone octamer (Figure 36-2). The majority of core histone proteins interact with the DNA on the inside of the supercoil without protruding, though the amino terminal tails of all the histones probably protrude outside of this structure and are available for regulatory covalent modifications (see Table 36-1). 

Figure 36-2. Model for the structure of the nucleosome, in which DNA is wrapped around the surface of a flat protein cylinder consisting of two each of histones H2A, H2B, H3, and H4 that form the histone octamer. The 146 base pairs of DNA, consisting of 1.75 superhelical turns, are in contact with the histone octamer. This protects the DNA from digestion by a nuclease. The position of histone HI, when it is present, is indicated by the dashed outline at the bottom of the figure.

Much of the DNA is associated with histone proteins to form a structure called the nucleosome. Nucleo-somes are composed of an octamer of histones and 150 bp of DNA. 

In the nuclei of all eukaryotic cells, DNA is tightly wrapped around an octamer of histone proteins and is compacted into a dense structure known as chromatin. In order to access the genetic information which is required in numerous essential cellular processes including DNA replication, gene expression and DNA repair, chromatin needs to be partially unwound. One important mechanism to regulate chromatin structure and thus to control the access of the genomic DNA is through histone modifications [1-6]. The histone octamer is composed of two copies of H2A, H2B, H3 and H4 core histone proteins. Their tails, that protrude out of the surface of the... 

The Beato group has studied in depth the influence of the nucleosome structure in response to glucocorticoids (Beato 1989). Nucleosomes are formed by segments of 120 nucleotides of the double helix of DNA that make two twists around an octamer of histone. There are 200 nucleotides between two consecutive nucleosomes, so that a gene normally has tens of nucleosomes. 

The nucleosome is composed of 200 base pairs of DNA and an octamer of the histones H2A, H2B, H3, and H4 as well as histone HI (Komberg, 1974, 1977). Nucleosomes can be obtained by mild digestion of chromatin with micrococcal nuclease (Noll, 1974a Axel, 1975), followed by fractionation on a sucrose gradient. Further digestion of the nucleosomes results in the formation of nucleosome core particles composed of 145 base pairs of DNA and an octamer of the histones H2A, H2B, H3, and H4 (Rill and Van Holde, 1973 Sollner-Webb and Felsenfeld, 1975 Axel, 1975 Bakayev et al., 1975 Whitlock and Simpson, 1976 Noll and Komberg, 1977). The DNA piece thus excised is called linker DNA which serves as a link... 

Hyde and Walker, 1975a) indicate that there are two basic types of histone-histone interactions or contacts those within the nucleosome which result in the formation of histone octamers and those between nucleosomes which give rise to higher oligomers. 

Cross-linking of chromatin in 2 M NaCl at pH 8.0 results in the formation of a cross-linked octamer (Thomas and Komberg, 1975a) which contains the four core histones in equimolar ratios (Thomas and Komberg, 1975b). A non-cross-linked complex of histones isolated in 2 M NaCl at pH 7.0 also was found to contain an equimolar ratio of the four core histones but had a molecular weight determined to be near that... 

The histone octamer of nucleosome core particles was cross-linked by dimethylsuberimidate and isolated from the DNA by precipitation in 3 M NaCl (0.05 M sodium phosphate buffer, pH 7.0). The cross-linked octamer, dissolved at low ionic strength, was reconstituted by mixing with DNA at 1.0 M NaCl (pH 8.0 Tris buffer) and dialyzed against 0.6 M NaCl in the same buffer. The reconstituted particle had properties similar to those of the cross-linked core particle. It sedi-... 

Similar results were obtained from reconstitution experiments with DNA and a non-cross-linked octamer (Thomas and Butler, 1978). Nucleosome-like particles were observed in the EM and a pattern of histone cross-linking comparable to that of native chromatin was obtained. However, only 140-base-pair repeats were obtained upon micrococcal nuclease digestion instead of 200-base-pair repeats obtained for native rat liver chromatin (Noll and Komberg, 1977). This indicates that, in the absence of HI, only core particles can be reconstituted. Nevertheless, these studies with both cross-linked and reassembled un-cross-linked histones demonstrate that the octamer is a complete biological functional unit retaining the information for folding the DNA around the histone core. 

As noted above, salt-extracted H2A H2B will form higher oligomers, fibers could be obtained from salt-extracted histones (Fig. 3c), and even octamers are now known to associate into superstructures at very high salt concentration (Wachtel and Sperling, 1979). It would therefore seem that self-assembly is a general property of histones. The pattern of complex formation of the core histones is shown schematically in Fig. 4. 

Upon increase in salt concentration to 2 M, histone octamers were obtained (Thomas and Butler, 1978). The octamer could be assembled from acid-extracted as well as from salt-extracted histones (Thomas and Butler, 1978). In a concentrated solution of the four core histones (prepared by acid extraction) at an ionic strength higher than 2 M NaCl (minimum 10 mg/ml histone concentration), there is a small fraction of assembled fibrous structures which can be observed in the electron microscope (Sperling and Bustin, 1976 Wachtel and Sperling, 1979). These fibers (see Fig. 3d) are 60 A in diameter and have a 330 A axial repeat, and were shown to be composed of the four core histones in an equimolar ratio (Wachtel and Sperling, 1979). The percentage of fibers in the solution of the four core histones is promoted by increase in histone and salt concentrations. 

Two types of subnucleosomal particles which retain many, if not all, of the properties of the intact nucleosome have been identified. The first type contains only H3 and H4, either as a tetramer (Bina-Stein and Simpson, 1977) or an octamer (Simon et al., 1978 Stockley and Thomas, 1979), while the second contains all core histones, each lacking up to 30 amino-terminal residues which have been digested away by trypsin (Whitlock and Simpson, 1977). The fact that other subnucleosomal particles have not been isolated does not necessarily mean that they cannot exist it indicates only that the proper reconstitution or dissociation conditions have not been found. Nevertheless, results to date point to H3-H4 on the one hand, and the trypsin-resistant carboxy-terminal regions of all the core histones on the other hand, as playing controlling structural roles in the formation of the nucleosome and the consequent folding of the DNA. 

As to the stoichiometry of the H3-H4-DNA particle, two complexes were identified an H3-H4 tetramer and an H3-H4 octamer, each associated with about 140 base pairs of DNA. The complexing of 140 base pairs of DNA with H3 and H4 resulted in the formation of nucleosome-like particles, as observed by the EM, and reported to have an s20base pairs (Bina-Stein and Simpson, 1977 Bina-Stein, 1978). These results differ from those of Simon et al. (1978) who report that at least two complexes of H3 H4-DNA can be obtained upon reconstitution of H3, H4, and 150 bp DNA. In this experiment both an octamer and a tetramer of H3-H4 were found bound to 150 base pairs of DNA, having sM,w equal to 10.4 and 7.5 for the octamer and tetramer, respectively. The stoichiometry of the complexes obtained is dependent on the histone-to-DNA ratio. At low ratios of histone to DNA the predominant species contains an H3-H4 tetramer per 150 base pairs of DNA. At a histone-to-DNA ratio of 1 1 the octamer prevails. The nuclease and protease digestion experiments (Camerini-Otero et al., 1976 Sollner-Webb et al., 1976) were performed at a histone-to-DNA ratio of 0.5, conditions which for 140-base-pair DNA would lead primarily to a tetrameric complex. Therefore, it seems that a tetramer of H3 H4 is sufficient for the generation of nuclease-resistant fragments similar to those of complete nucleosomes. Upon addition of H2A and H2B to the tetrameric complex, nucleosomes are formed. Addition of H3-H4 to the tetrameric complex resulted in an octameric complex which is similar in compaction to nucleosomes. H3-H4 tetramers and octamers were similarly found complexed with about 140 base pairs of DNA upon reconstitution of H3-H4 with SV40 DNA. Both complexes were reported to be able to fold 140 base pairs of DNA (Thomas and Oudet, 1979). 

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