Effect of in vivo Histone Hyperacetylation on the State of Chromatin Fibers

Abstract

We evaluated the contribution of in vivo histone acetylation to the folding of chromatin into its higher-order structures. We have compared high-order folding patterns of hyperacetylat- ed vs. unmodified chromatin in living green monkey kidney cells (CV1 line) using intercalator chloroquine diphospate to induce alterations in the twist of internucleosomal linker DNA. We have shown that histone hyperacetylation induced by antibiotic Trichostatin A significantly alters intercalator-mediated chromatin folding pattern.     

Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in?vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.     

Chromatin fiber folding: requirement for the histone H4 N-terminal tail

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.     

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.     

DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues

Histone chaperones are a diverse class of proteins that facilitate chromatin assembly. Their ability to stabilize highly abundant histone proteins in the cellular environment prevents non-specific interactions and promotes nucleosome formation, but the various mechanisms for doing so are not well understood. We now focus on the dynamic features of the DAXX histone chaperone that have been elusive from previous structural studies. Using hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS), we elucidate the concerted binding-folding of DAXX with histone variants H3.3/H4 and H3.2/H4 and find that high local stability at the variant-specific recognition residues rationalizes its known selectivity for H3.3. We show that the DAXX histone binding domain is largely disordered in solution and that formation of the H3.3/H4/DAXX complex induces folding and dramatic global stabilization of both histone and chaperone. Thus, DAXX uses a novel strategy as a molecular chaperone that paradoxically couples its own folding to substrate recognition and binding. Further, we propose a model for the chromatin assembly reaction it mediates, including a stepwise folding pathway that helps explain the fidelity of DAXX in associating with the H3.3 variant, despite an extensive and nearly identical binding surface on its counterparts, H3.1 and H3.2.     

Toward convergence of experimental studies and theoretical modeling of the chromatin fiber

Understanding the structural organization of eukaryotic chromatin and its control of gene expression represents one of the most fundamental and open challenges in modern biology. Recent experimental advances have revealed important characteristics of chromatin in response to changes in external conditions and histone composition, such as the conformational complexity of linker DNA and histone tail domains upon compact folding of the fiber. In addition, modeling studies based on high-resolution nucleosome models have helped explain the conformational features of chromatin structural elements and their interactions in terms of chromatin fiber models. This minireview discusses recent progress and evidence supporting structural heterogeneity in chromatin fibers, reconciling apparently contradictory fiber models.     

Structure of the H1 C-terminal domain and function in chromatin condensation

Linker histones are multifunctional proteins that are involved in a myriad of processes ranging from stabilizing the folding and condensation of chromatin to playing a direct role in regulating gene expression. However, how this class of enigmatic proteins binds in chromatin and accomplishes these functions remains unclear. Here we review data regarding the H1 structure and function in chromatin, with special emphasis on the C-terminal domain (CTD), which typically encompasses approximately half of the mass of the linker histone and includes a large excess of positively charged residues. Owing to its amino acid composition, the CTD was previously proposed to function in chromatin as an unstructured polycation. However, structural studies have shown that the CTD adopts detectable secondary structure when interacting with DNA and macromolecular crowding agents. We describe classic and recent experiments defining the function of this domain in chromatin folding and emerging data indicating that the function of this protein may be linked to intrinsic disorder.     

The Drosophila Df31 protein interacts with histone H3 tails and promotes chromatin bridging in vitro

Df31 is a small hydrophilic protein from Drosophila melanogaster that can act as a histone chaperone in vitro. The protein is also detected as an integral component of chromatin, present at approximately the same level as histone H1. We have developed a simple assay to measure protein binding to oligonucleosomes and used it to characterise the DF31-oligonucleosome interaction. DF31 bound to chromatin in vitro at a level comparable to that observed in vivo. The DF31-chromatin interaction required the presence of core histone tails but binding was independent of the presence of H1 in the chromatin. Multiple regions of DF31 contributed to the interaction. Df31-chromatin binding still occurred on chromatin containing only H3/4, and cross-linking experiments showed that Df31 made intimate contact with H3, suggesting that this might be the primary contact site. Finally, using immobilised chromatin templates, we showed that DF31 promoted interstrand bridging between two independent oligonucleosome chains. These results provide strong evidence for a structural role of DF31 in chromatin folding and give an indication of the mechanism involved.     

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 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.     

Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.     

Linker DNA bending induced by the core histones of chromatin

We have previously reported that ionic conditions that stabilize the folding of long chromatin into 30-nm filaments cause linker DNA to bend, bringing the two nucleosomes of a dinucleosome into contact [Yao, J., Lowary, P. T., & Widom, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7603-7607]. Dinucleosomes are studied because they allow the unambiguous detection of linker DNA bending through measurement of their nucleosome-nucleosome distance. Because of the large resistance of DNA to bending, the observed compaction must be facilitated by the histones. We have now tested the role of histone H1 (and its variant, H5) in this process. We find that dinucleosomes from which the H1 and H5 have been removed are able to compact to the same extent as native dinucleosomes; the transition is shifted to higher salt concentrations. We conclude that histone H1 is not essential for compacting the chromatin filament. However, H1 contributes to the free energy of compaction, and so it may select a single, ordered, compact state (the 30-nm filament, in long chromatin) from a family of compact states which are possible in its absence.