Nucleosome reconstitution

During nucleosome reconstitution, performed by mixing core histones and DNA in 2 M NaCl with slow back-dialysis to low salt con-... 

In order to investigate the structural and functional characteristics of the chromatin fiber, several methods for in vitro nucleosome reconstitution have been developed (Lusser and Kadonga, 2004). Among them, the salt-dialysis method is the simplest... 

Yoda K, Ando S, Morishita S, Houmura K, Hashimoto K, Takeyasu K, Okazaki T (2000) Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc Natl Acad Sci U S A 97 7266-7271... 

Fig. 3. Gallery of representative nucleosomes reconstituted in the absence (a) or presence of GH5 (b) or H5 (c), and visualized by scanning transmission electron microscopy, (a) and (b) 256 bp 5S rDNA fragment [65]. (c) 357 bp fragment from the 5S series (see text). Samples were diluted in TE buffer supplemented with 50 mM NaCl and 5 mM MgCl2 before adsorption to the grids. Note the nucleosome different positions relative to the DNA ends. Bars 25 nm and 75 bp. (Adapted from Fig. 7,9, and 10 in Ref [34].) Schemes of the corresponding DNA conformations are shown.

In the absence of conclusive data on the role of a positive supercoiling wave, static positive supercoiling elicited by nucleosome reconstitution on relaxed or slightly positively-supercoiled plasmids [51] or by ethidium bromide intercalation in the loop of mononucleosomes on DNA minicircles [52] did not succeed either in releasing dimers. Moreover, circular dichroism, histone chemical modi-flcation and H3-thiol accessibility failed to detect an even slight alteration in the structure of such torsionally-stressed nucleosomes [51]. The reason was later found to lie in the ability of nucleosome entry/exit DNAs to form a positive crossing [52]. 

Several lines of evidence indicate that CENP-A replaces conventional H3 in the nucleosome. Biochemical studies showed that CENP-A co-sediments with nucleo-some core particles [7] and a genetic analysis indicates an interaction between Cse4p, the CENP-A of Saccharomyces cerevisiae, and H4 [16,17]. A recent study with CENP-A purified from HeLa cells or expressed in bacteria showed that it can substitute for conventional H3 in nucleosome reconstitution [18]. Reconstituted CENP-A-containing nucleosomes appear to contain the other core histones in appropriate stoichiometry. However, they did not strongly protect 146 bp of core DNA from micrococcal nuclease, suggesting that CENP-A may significantly alter some aspects of the core nucleosome structure. 

All this leads to the question of how DNA affects nucleosomal stability at the molecular level. DNA bendability has been repeatedly put forward as a candidate to play an important role [280-282]. This property is related to the persistence length of DNA [283]. A study on the characterization of nucleosomes reconstituted onto methylated DNA [poly(dG-m dC) poly (dG-m dC)] [284] provides support to this hypothesis. Poly(dG-m dC) poly (dG-m dQ DNA can be induced to change from its B to its Z conformation in the presence of millimolar amounts of divalent ions [285] such as MgCl2. The persistence length of poly(dG-m dC) poly (dG-m dC) in the Z form in high salt was found to be 208 nm (ca. 612 bp) [286], a value much lower than that of the same polymer in the B form (93.8 nm, 276 bp) at... 

In the early work on this topic, Kleinschmidt and Martinson [219] used reconstituted nucleosome core particles consisting of two uH2A molecules to find that these particles had structural features that were almost indistinguishable from those of particles reconstituted with native histones or from native nucleosome core particles. A more exhaustive and detailed study in the same topic carried out a few years later [220] came in corroboration of these results and showed that the same observations could be extended to nucleosomes reconstituted with uH2B. Furthermore, the NaCl dependent stability of the uH2A reconstituted nucleosome core particles is identical to that exhibited by the native counterpart [222]. 

Figure 5-21 Nucleosomes. (A) Electron micrographs of individual nucleosomes reconstituted from 256-bp DNA fragments and separated proteins. From Hamiche et al.213 Courtesy of Ariel Prunell. (B) Model of a nucleosome core. The 1.75-tum (145-bp) DNA superhelix winds around the histone octomer which consists of two subunits apiece of histones H2A, H2B, H3, and H4. In addition, two elongated molecules of proteins HMG-14 or HMG-17 are indicated (see also Chapter 27). (C) Schematic radial projection of the doublehelical DNA showing areas protected from cleavage by hydroxyl radicals (see Fig. 5-50) by the bound proteins. The shaded areas are those protected by HMGs. The zigzag lines near the dyad axis indicate the most prominent regions of protection. (B) and (C) are from Alfonso et al.2U...

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. 

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. 

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). 

Since 1974, evidence has accumulated in the literature which indicates that chromatin itself may be considered as an assembly system. It is true that chromatin is more complex than assembly systems analyzed to date, both with respect to the size of the nucleic acid involved and therefore the amount (and variety) of protein complexed with it and with respect to the dynamic aspect of the multilevel higher order structure. Nevertheless, at least at the lower levels of organization, the interpretation of chromatin as an assembly system may be valid. Evidence for this derives from three basic lines of research described in previous sections (1) the reconstitution of the nucleosome, (2) the self-assembly of the octamer, and (3) the putative self-organization of nucleosomes into higher order structures. 

The fact that nucleosome-like particles can be reversibly reconstituted from histones and DNA, without any additional factor, exemplifies the principle of self-assembly of biological structures. The reconstituted particles have the characteristic beaded appearance of nu-... 

The effect of the superhelical strain of the DNA template on the nucleosome structure can be investigated from the in vitro chromatin reconstitution system (for the detail of in vitro chromatin reconstitution, see sections 2.1 and 2.3). Interestingly, the efficiency of the reconstitution becomes higher as the lengths of the DNA used are longer (Hizume et al, 2004) (Fig. 3a-c). In the 3 kb reconstituted chromatin, one nucleosome could be formed in every 826 bp DNA on average, while in the 106 kb chromatin fibers, one nucleosome can be formed in every 260 bp of DNA. The chromatin reconstituted on the any length of linearized plasmid, the efficiency of the reconstitution becomes one nucleosome per 800 bp DNA. The treatment of the... 

Figure 3. The stability of the nucleosome is affected by the length and the superhelicity of DNA. (a-b) The chromatin fibers were reconstituted from the purified plasmids and the histone octamers by a salt-dialysis method and observed under AFM. The 3 kb (a) or 106 kb (e) supercoiled circular plasmid was used as a template, (c) Relationship between the plasmid length and the frequency of nucleosome formation in the reconstitution process. The nucleosome frequency is represented as the number of base pairs per nucleosome and plotted against the length of the template DNA in supercoiled (filled circle) and linear (open circle) forms, (d) AFM image of the chromatin fiber reconstituted on the topoisomerase 1-treated plasmid, (e) Chromatin fiber reconstituted with Drosophila embryo extract. The chromatin fiber was reconstituted from plasmid DNA of 10kband the embryo extract of Drosophila, and was observed by AFM...

Biochemical reconstitution of the 30 nm fiber has recently been succeeded by using a salt-dialysis procedure with a long DNA template (>100 kb) (Hizume et al, 2005). AFM imaging of the reconstituted chromatin has shown that the beads-on-a-string structure of the nucleosomes ( 400 nucleosomes on 100 kb DNA) are converted to a thicker fiber in the presence of histone HI. The thickness of the fiber changes reversibly between 20 nm and 30 nm, depending on the salt environment (in 50 mM and 100 mM NaCl, respectively) (Fig. 4) namely, the linker histone directly promotes a thicker fiber formation in a salt-dependent manner. 

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