Detection of interactions between nucleosome arrays mediated by specific core histone tail domains

The core histone tail domains play important roles in different stages of chromatin condensation. The tails are required for folding nucleosome arrays into secondary chromatin structures such as the approximately 30 nm diameter chromatin fiber and for mediating fiber-fiber interactions important for formation of tertiary chromatin structures. Crosslinking studies have demonstrated that inter-nucleosomal tail-DNA contacts appear in conjunction with salt-induced folding of nucleosome arrays into in higher order chromatin structures. However, since both folding of nucleosome arrays and fiber-fiber interactions take place simultaneously in >2-3 mM MgCl(2) such inter-nucleosome interactions may reflect short range (intra-array) or longer range (inter-array) interactions. Here, we describe a novel technique to specifically identify inter-array interactions mediated by the histone tail domains. In addition, we describe a new method for the preparation of H3/H4 tetramers.     

A Distinct Switch in Interactions of the Histone H4 Tail Domain upon Salt-dependent Folding of Nucleosome Arrays

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes (1, 2). Likewise, H4 tail-H2A contacts stabilize array folding (3). However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.     

Salt-dependent intra- and internucleosomal interactions of the H3 tail domain in a model oligonucleosomal array

The core histone tail domains are known to be key regulators of chromatin structure and function. The tails are required for condensation of nucleosome arrays into secondary and tertiary chromatin structures, yet little is known regarding tail structures or sites of tail interactions in chromatin. We have developed a system to test the hypothesis that the tails participate in internucleosomal interactions during salt-dependent chromatin condensation, and here we used it to examine interactions of the H3 tail domain. We found that the H3 tail participates primarily in intranucleosome interactions when the nucleosome array exists in an extended "beads-on-a-string" conformation and that tail interactions reorganize to engage in primarily internucleosome interactions as the array successively undergoes salt-dependent folding and oligomerization. These results indicated that the location and interactions of the H3 tail domain are dependent upon the degree of condensation of the nucleosomal array, suggesting a mechanism by which alterations in tail interactions may elaborate different structural and functional states of chromatin.     

Intra- and inter-nucleosome interactions of the core histone tail domains in higher-order chromatin structure

Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array–array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, effect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as inter-nucleosomal and inter-array interactions of the H3 and H4 N-terminal tails.     

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.     

Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg²⁺, Ca²⁺, cobalt(III)hexammine³⁺, spermidine³⁺ and spermine⁴⁺] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K → Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.     

The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na(+), K(+), Mg(2+), Ca(2+), cobalt-hexammine(3+), spermidine(3+) and spermine(4+). Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K(+), (as well as Rb(+) and Cs(+)) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K(+) (and Rb(+)/Cs(+)) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.     

Intra- and inter-nucleosomal protein-DNA interactions of the core histone tail domains in a model system

The core histone tail domains are key regulators of eukaryotic chromatin structure and function and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here we introduce a method for examining inter-nucleosome interactions of the tail domains in a model dinucleosome and determine the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, we reconstituted a nucleosome containing a single histone site specifically modified with a photoinducible cross-linker within the histone tail domain, and a second nucleosome containing a radiolabeled DNA template. These two nucleosomes were then ligated together and cross-linking induced by brief UV irradiation under various solution conditions. After cross-linking, the two templates were again separated so that cross-linking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal cross-links. Our results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Additionally, we find that binding of a linker histone to the dinucleosome increased the association of the H3 and H4 tails with the linker DNA region.     

Structures and interactions of the core histone tail domains

The core histone tail domains are "master control switches" that help define the structural and functional characteristics of chromatin at many levels. The tails modulate DNA accessibility within the nucleosome, are essential for stable folding of oligonucleosome arrays into condensed chromatin fibers, and are important for fiber-fiber interactions involved in higher order structures. Many nuclear signaling pathways impinge upon the tail domains, resulting in posttranslational modifications that are likely to alter the charge, structure, and/or interactions of the core histone tails or to serve as targets for the binding of ancillary proteins or other enzymatic functions. However, currently we have only a marginal understanding of the molecular details of core histone tail conformations and contacts. Here we review data related to the structures and interactions of the core histone tail domains and how these domains and posttranslational modifications therein may define the structure and function of chromatin.     

The H3 tail domain participates in multiple interactions during folding and self-association of nucleosome arrays

The core histone tail domains play a central role in chromatin structure and epigenetic processes controlling gene expression. Although little is known regarding the molecular details of tail interactions, it is likely that they participate in both short-range and long-range interactions between nucleosomes. Previously, we demonstrated that the H3 tail domain participates in internucleosome interactions during MgCl(2)-dependent condensation of model nucleosome arrays. However, these studies did not distinguish whether these internucleosome interactions represented short-range intra-array or longer-range interarray interactions. To better understand the complex interactions of the H3 tail domain during chromatin condensation, we have developed a new site-directed cross-linking method to identify and quantify interarray interactions mediated by histone tail domains. Interarray cross-linking was undetectable under salt conditions that induced only local folding, but was detected concomitant with salt-dependent interarray oligomerization at higher MgCl(2) concentrations. Interestingly, lysine-to-glutamine mutations in the H3 tail domain to mimic acetylation resulted in little or no reduction in interarray cross-linking. In contrast, binding of a linker histone caused a much greater enhancement of interarray interactions for unmodified H3 tails compared to "acetylated" H3 tails. Collectively these results indicate that H3 tail domain performs multiple functions during chromatin condensation via distinct molecular interactions that can be differentially regulated by acetylation or binding of linker histones.     

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.     

30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.     

Clipping of Flexible Tails of Histones H3 and H4 Affects the Structure and?Dynamics of the Nucleosome

Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail’s lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA.     

Histone octamer instability under single molecule experiment conditions

We have studied the sample concentration-dependent and external stress-dependent stability of native and reconstituted nucleosomal arrays. Whereas upon stretching a single chromatin fiber in a solution of very low chromatin concentration the statistical distribution of DNA length released upon nucleosome unfolding shows only one population centered around approximately 25 nm, in nucleosome stabilizing conditions a second population with average length of approximately 50 nm was observed. Using radioactively labeled histone H3 and H2B, we demonstrate that upon lowering the chromatin concentration to very low values, first the linker histones are released, followed by the H2A-H2B dimer, whereas the H3-H4 tetramer remains stably attached to DNA even at the lowest concentration studied. The nucleosomal arrays reconstituted on a 5 S rDNA tandem repeat exhibited similar behavior. This suggests that the 25-nm disruption length is a consequence of the histone H2A-H2B dimer dissociation from the histone octamer. In nucleosome stabilizing conditions, a full approximately 145 bp is constrained in the nucleosome. Our data demonstrate that the nucleosome stability and histone octamer integrity can be severely degraded in experiments where the sample concentration is low.