The H4 Tail Domain Participates in Intra- and Internucleosome Interactions with Protein and DNA during Folding and Oligomerization of Nucleosome Arrays

ABSTRACT The condensation of nucleosome arrays into higher-order secondary and tertiary chromatin structures likely involves long-range internucleosomal interactions mediated by the core histone tail domains. We have characterized interarray interactions mediated by the H4 tail domain, known to play a predominant role in the formation of such structures. We find that the N-terminal end of the H4 tail mediates interarray contacts with DNA during self-association of oligonucleosome arrays similar to that found previously for the H3 tail domain. However, a site near the histone fold domain of H4 participates in a distinct set of interactions, contacting both DNA and H2A in condensed structures. Moreover, we also find that H4-H2A interactions occur via an intra- as well as an internucleosomal fashion, supporting an additional intranucleosomal function for the tail. Interestingly, acetylation of the H4 tail has little effect on interarray interactions by itself but overrides the strong stimulation of interarray interactions induced by linker histones. Our results indicate that the H4 tail facilitates secondary and tertiary chromatin structure formation via a complex array of potentially exclusive interactions that are distinct from those of the H3 tail domain.

Acetylation Mimics within Individual Core Histone Tail Domains Indicate Distinct Roles in Regulating the Stability of Higher-Order Chromatin Structure

ABSTRACT Nucleosome arrays undergo salt-dependent self-association into large oligomers in a process thought to recapitulate essential aspects of higher-order tertiary chromatin structure formation. Lysine acetylation within the core histone tail domains inhibits self-association, an effect likely related to its role in facilitating transcription. As acetylation of specific tail domains may encode distinct functions, we investigated biochemical and self-association properties of model nucleosome arrays containing combinations of native and mutant core histones with lysine-to-glutamine substitutions to mimic acetylation. Acetylation mimics within the tail domains of H2B and H4 caused the largest inhibition of array self-association, while modification of the H3 tail uniquely affected the stability of DNA wrapping within individual nucleosomes. In addition, the effect of acetylation mimics on array self-association is inconsistent with a simple charge neutralization mechanism. For example, acetylation mimics within the H2A tail can have either a positive or negative effect on self-association, dependent upon the acetylation state of the other tails and nucleosomal repeat length. Finally, we demonstrate that glutamine substitutions and lysine acetylation within the H4 tail domain have identical effects on nucleosome array self-association. Our results indicate that acetylation of specific tail domains plays distinct roles in the regulation of chromatin structure.

The H3 Tail Domain Participates in Multiple Interactions during Folding and Self-Association of Nucleosome Arrays

ABSTRACT 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 MgCl2-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 MgCl2 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.

Physical methods used to study core histone tail structures and interactions in solutionThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

The core histone tail domains are key regulatory elements in chromatin. The tails are essential for folding oligonucleosomal arrays into both secondary and tertiary structures, and post-translational modifications within these domains can directly alter DNA accessibility. Unfortunately, there is little understanding of the structures and interactions of the core histone tail domains or how post-translational modifications within the tails may alter these interactions. Here we review NMR, thermal denaturation, cross-linking, and other selected solution methods used to define the general structures and binding behavior of the tail domains in various chromatin environments. All of these methods indicate that the tail domains bind primarily electrostatically to sites within chromatin. The data also indicate that the tails adopt specific structures when bound to DNA and that tail structures and interactions are plastic, depending on the specific chromatin environment. In addition, post-translational modifications, such as acetylation, can directly alter histone tail structures and interactions.

The Core Histone N-Terminal Tail Domains Negatively Regulate Binding of Transcription Factor IIIA to a Nucleosome Containing a 5S RNA Gene via a Novel Mechanism

ABSTRACT Reconstitution of a DNA fragment containing a 5S RNA gene from Xenopus borealis into a nucleosome greatly restricts binding of the primary 5S transcription factor, TFIIIA. Consistent with transcription experiments using reconstituted templates, removal of the histone tail domains stimulates TFIIIA binding to the 5S nucleosome greater than 100-fold. However, we show that tail removal increases the probability of 5S DNA unwrapping from the core histone surface by only approximately fivefold. Moreover, using site-specific histone-to-DNA cross-linking, we show that TFIIIA binding neither induces nor requires nucleosome movement. Binding studies with COOH-terminal deletion mutants of TFIIIA and 5S nucleosomes reconstituted with native and tailless core histones indicate that the core histone tail domains play a direct role in restricting the binding of TFIIIA. Deletion of only the COOH-terminal transcription activation domain dramatically stimulates TFIIIA binding to the native nucleosome, while further C-terminal deletions or removal of the tail domains does not lead to further increases in TFIIIA binding. We conclude that the unmodified core histone tail domains directly negatively influence TFIIIA binding to the nucleosome in a manner that requires the C-terminal transcription activation domain of TFIIIA. Our data suggest an additional mechanism by which the core histone tail domains regulate the binding of trans-acting factors in chromatin.