The histone fold: evolutionary questions.

While histones as a class of protein associated with DNA have been known for over a hundred years, our current ideas of how histones associate with DNA began with the discovery of the nucleosome as a basic structural unit of chromatin (1, 2). In the nucleosome, two left-handed superhelical turns of DNA wrap around an octameric histone core consisting of two copies of each of the four core histonesH2A, H2B, H3, and H4. The publication by Moudrianakis and coworkers of the crystal structure of the histone octamer (3) was a landmark in the field of chromatin. An immediate reason was that the crystal structure was in good agreement with previous electron-microscopic studies on the octamer (4), as well as with the crystal structure of the nucleosome core particle at 7 A (5), thereby resolving an earlier controversy. The surface of the octamer had a left-handed helical ramp to which DNA presumably was bound in the nucleosome. When DNA was modeled onto this ramp, it agreed very well with the general features of the DNA observed in the nucleosome core particle (5). This showed that the structure of the isolated octamer was likely to be similar to its structure in the nucleosome, thus establishing the relevance of the work for chromatin. However, the crystal structure of the octamer was important for a more fundamental reason: it was the first structure at sufficient resolution to allow the tertiary fold of the individual core histones to be determined, with some surprising results. It has been known for a long time that histones associate with one another in solution. The most stable interactions are those of the heterotypic dimers H2A-H2B and H3-H4 (6). The H3-H4 dimers further associate to form a stable (H3-H4)2 tetramer. The crystal structure of the octamer shows that the histones are indeed associated as heterotypic dimers within the octamer and revealed the basis for this dimerization: despite little sequence similarity, all four histones share a common structural motif, the histone fold, which is involved in dimerization with its partner. As shown in Fig. 1 for the H3-H4 dimer, each histone is highly elongated and clasps its partner in the dimer by means of extensive interactions in what Arents et al. (3) term a "handshake motif." Such extensive interactions between the members of each dimer pair had been suggested by NMR studies (7, 8). In a recent issue of the Proceedings (9), Arents and Moudrianakis use the structure of the core histones to investigate the chemical and structural nature of the histone fold and offer arguments for a common origin for the core histones. In a related paper (10), it is shown that the histone fold occurs in a variety of different proteins. Taken together, these results show that the fold forms the basis for a distinct family of proteins. Each of the core histones is highly conserved (11, 12). Histones H3 and H4 are among the most highly conserved proteins known, both in sequence and in length. Histones H2A and H2B have regions that are more variable, especially at their termini, but each has a highly conserved core. However, the four core histones have little sequence similarity with respect to one another, with pairwise identities that range from 15% to 20%. Despite the low sequence identity, a common origin for the core histones had been suggested by comparisons of the core histone amino acid sequences (13, 14). These studies suggested that the core histones were related to one another, but not to the linker histone Hi. Further, both studies offered evidence that histone H2A had an internal repeat that suggested that it was the product of a gene doubling event. However, a study that compared gene sequences questioned the gene doubling of H2A (15), and there were some inconsistencies in the conclusions of the three studies, perhaps because of the limited number of complete histone sequences available at the time as well as the low degree of sequence identity. Because of these inconsistencies, these studies were not regarded as definitive [see discussion by van Holde (16)]. Nevertheless, gene doubling in H2A was also suggested by van Holde (16), who showed a sequence similarity between the N-terminal region of the archaebacterial DNA-binding protein HTa with both the Nand C-terminal domains of H2A. It now turns out that the basic conclusions of these sequence comparison studies, although incomplete, were right. Subsequent analysis showed that the histone fold itself could be considered as a repeat of a basic helix-strand-helix (HSH) motif that occurred in both halves of the fold (17); this suggests that the long central helix in the histone fold arose as a result of a fusion between helices from each HSH motif. Since the basic structural unit is half a fold, this lends support to the idea of a gene-doubling in the histones, although it is clearly present in all the histones and not just H2A as suggested by sequence comparison studies. Moreover, this suggests that the gene-doubling event occurred before the differentiation of the core histones into the four types. Arents and Moudrianakis (9) have now made quantitative comparisons of the degree of structural similarity between the histone folds of the four core histones. These comparisons show that the fold is quite similar in the four histones. If the Nterminal and C-terminal halves of the histone fold (or just the HSH motif) are compared, the similarity is even greater, but this is not unexpected because the motif being compared is twice as small and may have fewer degrees of freedom structurally. Apart from the structural comparisons, other arguments are offered for a common origin for the core histones. Within each dimer, the fold portions of the histones are related by an approximate two-fold axis (see Fig. 1). This suggests that, although today the histone dimers are heterotypic, the ancestral dimer may have been a homodimer with a true twofold axis. More importantly, the point is made that histone-like proteins in archaebacteria are more similar to each of the core histones than any one of the core histones is to the others. It is not difficult to rationalize why each core histone has been highly conserved. Each histone has to make numerous interactions, including interactions with its dimer partner, with other components of the octamer, and with DNA, and is therefore subject to a variety of selective pressures. However, this in itself creates another puzzle. If the core histones descended from a common ancestor, it is clear that they have diverged considerably over time into the four types. If they were allowed to diverge in this manner, what slowed the divergence once the four core histones had differentiated? Although arguments have been made for why the core histones have their particular structure to facilitate the formation of the octamer and the nucleosome (3), it is not clear that four different subtypes are needed to form nucleosome-like struc-