H3/H4 tetramer

Specifically, when histones are dissociated from DNA in 2 M NaCl, and H3 H4 tetramer and H2A-H2B dimer may be identified after fractionation of the histones at pH 5.0 and cross-linking with dimethylsu-berimidate (Komberg and Thomas, 1974). The inference was drawn that these complexes exist as such in the intact nucleosome. Furthermore, since both the un-cross-linked H3 H4 tetramer, and the uncross-linked H2A-H2B dimer are stable complexes, it has proved possible to characterize their physical properties in solution. Some of these results are summarized here. 

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). 

FKBP H2A-H2B (dimer) H3-H4 (tetramer) Histone chaperone regulating rDNA silencing... 

Fig. 16. Cacodylate binding site between NCP molecules. Figure shows details of the stacking interactions between two neighboring molecules involving the dorsal H3 H4 tetramer face of one NCP and the ventral H2A H2B dimer face an adjacent NCP. The acidic residues forming a surface patch on the dimer face are rendered in ball-and-stick. The H4 tail is in blue and the H3 region involved in the interaction is in yellow. The cacodylate is shown as a CPK rendering.

Ubiquitinated histones have been suggested to destabilize the interface between the H2A-H2B dimers and the H3-H4 tetramer [211], and to be depleted from highly condensed mitotic chromosomes and enriched in HI deficient chromatin [212]. In Drosophila, the inducible hsp70 and copia genes are ubiquitinated, which represents... 

Duplicating the GFP-H2B experiments with both GFP-H3 and GFP-H4 vectors, very little H3 and H4 exchange outside of S phase was observed. Fluorescence of GFP-H3 and GFP H4 in HeLa cells in G1 recovered rapidly. The extremely rapid recovery rate is similar to that of a diffuse soluble protein, indicating that at this stage of the cell cycle, GFP-tagged H3 and -H4 are not incorporated into chromatin. FRAP experiments of transfected cells in S or G2 show that there is very little recovery of GFP-H3 or -H4 fluorescence. The fluorescence imaging indicates that once the H3 and H4 proteins are incorporated into the chromatin, they are essentially immobile for the remainder of the cell cycle. Unlike histones H2A and H2B, which associate as dimers in the nucleosome histone octamer, there is very little exchange of the components of the H3/H4 tetramer. 

The model of Fig. IB is taken from a review by van Holde et al. [3] which I refer to as the disruptive model. In this model the polymerase causes conditions (step A) which promote not only the displacement of the entry site H2A, H2B dimer from DNA, but also from the H3, H4 tetramer (step B). As a result of this disruption, the polymerase is free to transcribe through the tetramer alone without a general displacement from its associated DNA (step C). The H2A, H2B dimer is now free to reassociate to the vacated entry site (step D) to re-establish contacts with both the DNA and the H3, H4 tetramer. As transcription proceeds into the exit site H2A, H2B dimer, these proteins are now displaced from both the DNA and the H3, H4 tetramer in a similar manner as the entry site H2A, H2B dimer (step E). A positive feature with regard to this model is that by displacement of H2A, H2B, the polymerase is able to transcribe the DNA with half the histones displaced prior to transcription. Therefore both models, spooling and disruptive , describe mechanisms which would favorably enhance the process of transcription. Support for the disruptive model comes from the substantial in vivo information which suggests that nucleosomes undergo substantial disruption during transcription, as was described in the previous section. Of particular note are those observations which indicate that a discrete population of H2A, H2B... 

In (b) we see this histone octamer inserted into the nucleosome core. The H3-H4 tetramer is shown in yellow, and an H2a-H2b dimer is shown at each end in purple. 

The proteins of chromatin are either histones or nonhistones. There are five different types of histones, HI, H2A, H2B, H3, and H4. HI is called the "linker" histone while the others are "core" histones. In chromatin, two H2A/H2B dimers and one H3/H4 tetramer form an octamer around which DNA is wound in about 1.7 turns of a supercoil, to give a nucleosome core particle. Addition of histone HI and more DNA to the core particle forms the nucleosome, the major repeating substructure of chromatin. Adjacent nucleosomes are linked by DNAthe linker DNAtypically 30 bp in length. 

Once the H3/H4 tetramer is formed, it is joined by two dimers of H2A/H2B, primarily via hydrophobic and hydrogen bond contacts between histone H4 with histone H2B. The resulting daisychain of histones can be represented in linear form to illuminate the pattern of histone-histone interactions (contacts made within each dimer are... 

---