DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal

Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed.     

Nucleosome structure and conformational changes.

We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests (as did earlier probe experiments) that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5-0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

Acetylated nucleosome assembly on telomeric DNAs

The role of histone N-terminal domains on the thermodynamic stability of nucleosomes assembled on several different telomeric DNAs as well as on 'average' sequence DNA and on strong nucleosome positioning sequences, has been studied by competitive reconstitution. We find that histone tails hyperacetylation favors nucleosome formation, in a similar extent for all the examined sequences. On the contrary, removal of histone terminal domains by selective trypsinization causes a decrease of nucleosome stability which is smaller for telomeres compared to the other sequences examined, suggesting that telomeric sequences have only minor interactions with histone tails. Micrococcal nuclease kinetics shows enhanced accessibility of acetylated nucleosomes formed both on telomeric and 'average' sequence DNAs. These results suggest a more complex role for histone acetylation than the decrease of electrostatic interactions between DNA and histones.     

Effects of core histone tail domains on the equilibrium constants for dynamic DNA site accessibility in nucleosomes

The N and C-terminal tail domains of the core histones play important roles in gene regulation, but the mechanisms through which they act are not known. These tail domains are highly positively charged and are the sites of numerous post-translational modifications, including many sites for lysine acetylation. Nucleosomes in which these tail domains have been removed by trypsin remain otherwise intact, and are used by many laboratories as a model system for highly acetylated nucleosomes. Here, we test the hypothesis that one role of the tail domains is to directly regulate the accessibility of nucleosomal DNA to other DNA-binding proteins. Three assays are used: equilibrium binding by a site-specific, DNA-binding protein, and dynamic accessibility to restriction enzymes or to a non-specific exonuclease. The effects of removal of the tail domains as monitored by each of these assays can be understood within the framework of the site exposure model for the dynamic equilibrium accessibility of target sites located within the nucleosomal DNA. Removal of the tail domains leads to a 1.5 to 14-fold increase in position-dependent equilibrium constants for site exposure. The smallness of the effect weighs against models for gene activation in which histone acetylation is a mandatory initial event, required to facilitate subsequent access of regulatory proteins to nucleosomal DNA target sites. Alternative roles for histone acetylation in gene regulation are discussed.     

Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI

The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.     

Unraveling linker histone interactions in nucleosomes

Considerable progress has been made recently in defining the interactions of linker histones (H1s) within nucleosomes. Major advancements include atomic resolution structures of the globular domain of full-length H1s in the context of nucleosomes containing full-length linker DNA. Although these studies have led to a detailed understanding of the interactions and dynamics of H1 globular domains in the canonical on-dyad nucleosome binding pocket, more information regarding the intrinsically disordered N-terminal and C-terminal domains is needed. In this review, we highlight studies supporting our current understanding of the structures and interactions of the N-terminal, globular, and C-terminal domains of linker histones within the nucleosome.     

The divalent cations Ca2+ and Mg2+ play specific roles in stabilizing histone-DNA interactions within nucleosomes that are partially redundant with the core histone tail domains

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational repositioning upon removal of the core histone tail domains under physiological conditions, indicating that the tails influence the choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a nonbiased chemical cleavage reagent, indicative of an increase in the motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg(2+) and Ca(2+) can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict repositioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg(2+) after tail removal, the original distribution of translational positions was not re-established, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn(2+), Fe(2+), Co(2+), and Mn(2+) had little or no effect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg(2+) and Ca(2+) ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains.