A mutational mimic analysis of histone H3 post-translational modifications: specific sites influence the conformational state of H3/H4, causing either positive or negative supercoiling of DNA.

Histone H3 has specific sites of post-translational modifications that serve as epigenetic signals to cellular machinery to direct various processes. Mutational mimics of these modifications (glutamine for acetylation, methionine and leucine for methylation, and glutamic acid for phosphorylation) were constructed at the relevant sites of the major histone variant, H3.2, and their effects on the conformational equilibrium of the H3/H4 tetramer at physiological ionic strength were determined when bound to or free of DNA. The deposition vehicle used for this analysis was NAP1, nucleosome assembly protein 1. Acetylation mimics in the N-terminus preferentially stabilized the left-handed conformer (DNA negatively supercoiled), and mutations within the globular region preferred the right-handed conformer (DNA positively supercoiled). The methylation mimics in the N-terminus tended to maintain characteristics similar to those of wild-type H3/H4; i.e., the conformational equilibrium maintains similar levels of both left- and right-handed conformers. Phosphorylation mimics facilitated a mixed effect, i.e., when at serines, the left-handed conformer, and at threonines, a mixture of both conformers. When double mutations were present, the conformational equilibrium was shifted dramatically, either leftward or rightward depending on the specific sites. In contrast, these mutations tended not to affect the direction and extent of supercoiling for variants H3.1 and H3.3. Variant H3.3 promoted only the left-handed conformer, and H3.1 tended to maintain both conformers. Additional experiments indicate the importance of a propagation mechanism for ensuring the formation of a particular superhelical state over an extended region of the DNA. The potential relevance of these results to the maintenance of epigenetic information on a gene is discussed.

The nucleosome family: dynamic and growing.

Ever since the discovery of the nucleosome in 1974, scientists have stumbled upon discrete particles in which DNA is wrapped around histone complexes of different stoichiometries: octasomes, hexasomes, tetrasomes, "split" half-nucleosomes, and, recently, bona fide hemisomes. Do all these particles exist in vivo? Under what conditions? What is their physiological significance in the complex DNA transactions in the eukaryotic nucleus? What are their dynamics? This review summarizes research spanning more than three decades and provides a new meaning to the term "nucleosome." The nucleosome can no longer be viewed as a single static entity: rather, it is a family of particles differing in their structural and dynamic properties, leading to different functionalities.

Acetylation of H4 suppresses the repressive effects of the N-termini of histones H3/H4 and facilitates the formation of positively coiled DNA.

We have studied the role of the N-termini of histones H3/H4 in the regulation of the conformational changes that occur in H3/H4 during their deposition on DNA by NAP1 (nucleosome assembly protein 1). Removal of the N-termini extensively increased the right-handed conformation of H3/H4 as assayed by the increased levels of positive coils that were formed on DNA. The osmolytes, TMAO, betaine, sarcosine, alanine, glycine, and proline to varying degrees, facilitated the formation of positive coils. The denaturant, urea (0.6 M), blocked the osmolyte effects, causing a preference of H3/H4 to form negative coils (the left-handed conformation). Acetylated H3/H4 also formed high levels of positive coils, and it is proposed that both the osmolytes and acetylation promote the formation of an alpha-helix in the N-termini. This structural change may ultimately explain a unique feature of transcription through nucleosomes, i.e., that H2A/H2B tends to be more mobile than H3/H4. By using combinations of H3 and H4 that were either acetylated or the N-termini removed, it was also determined that the N-terminus of H4 is primarily responsible for repressing the formation of positive coils. Additional gradient analyses indicate that NAP1 establishes an equilibrium with the H3/H4-DNA complexes. This equilibrium facilitates a histone saturation of the DNA, a unique state that promotes the right-handed conformation. NAP1 persists in the binding of the complexes through interaction with the N-terminus of H3, which may be a mechanism for subsequent remodeling of the nucleosome during transcription and replication.

NAP1 catalyzes the formation of either positive or negative supercoils on DNA on basis of the dimer-tetramer equilibrium of histones H3/H4.

We have studied the tetramer-dimer equilibrium of histones H3/H4 and its effect on DNA supercoiling. Two approaches were found to shift the equilibrium toward dimer. In both instances, when deposited on DNA, the dimers formed positively coiled DNA. The first approach was to modify cysteine 110 of H3 with 5,5'-dithio-bis(2-nitrobenzoic acid (DTNB) and to directly add the histones to DNA at physiological ionic strength. The second approach involved adding an excess of the histone chaperone, nucleosome assembly protein 1 (NAP1) to the H3/H4 prior to deposition on the DNA. It was also observed that when H3/H4 were deposited in the tetrameric state, negatively coiled DNA was formed. The topological state of the DNA prior to deposition was also found to influence the final conformational state of H3/H4. It is proposed that in the tetrameric state, the H3-H3 interface has a left-handed pitch prior to binding DNA. In the dimeric state, the H3-H3 interface is not established until bound to DNA, at which point either the left or right-handed pitch will form on the basis of the initial topology of the DNA. Formaldehyde cross-linking and reversal were applied to identify the histone-histone interactions that facilitate the formation of positive stress. Higher-order interactions between multiple H3/H4 dimers were required to propagate this specific conformation. Changes in the conformational state of H3/H4 were also observed when the histones were bound to DNA prior to treatment with NAP1. It is proposed that these conformational changes in H3/H4 are involved in promoter activation and transcription elongation through nucleosomes.

Urea-nuclease treatment of concentrated retrovirions preserves viral RNA and removes polymerase chain reaction-amplifiable cellular RNA and DNA.

Cellular nucleic acids can interfere with the molecular cloning of retroviruses, a problem that is particularly serious with viruses propagated in lymphoblastoid cells that release large amounts of microvesicles and other cellular components. The approach taken to circumvent such problems involved first suspending viral pellets in water to allow any residual microvesicles to swell and perhaps lyse during overnight or longer incubation periods. Urea was then added to a concentration of 1.5-2.0 M to uncoil proteins that may protect nucleic acids from hydrolysis on the further addition of Micrococcal nuclease and ribonuclease A, both of which remain enzymatically active in molar urea solutions. The viral RNA was extracted and residual DNA removed by deoxyribonuclease I treatments. The utility of the method was demonstrated with two different retroviruses, a Moloney murine leukemia virus variant and Rous sarcoma virus, such that viral RNA thus purified was shown to be free of contamination by PCR-amplifiable cellular GAPDH mRNA and ribosomal RNA. This general approach should be applicable to viruses of any type in circumstances where contamination by cellular RNA and DNA poses a problem.

The FK506-binding protein, Fpr4, is an acidic histone chaperone.

Fpr4, a FK506-binding protein (FKBP), is a recently identified novel histone chaperone. How it interacts with histones and facilitates their deposition onto DNA, however, are not understood. Here, we report a functional analysis that shows Fpr4 forms complexes with histones and facilitates nucleosome assembly like previously characterized acidic histone chaperones. We also show that the chaperone activity of Fpr4 resides solely in an acidic domain, while the peptidylprolyl isomerase domain conserved among all FKBPs inhibits the chaperone activity. These observations argue that Fpr4, while unique structurally, deposits histones onto DNA for nucleosome assembly through the well-established mechanism shared by other chaperones.

Histone release during transcription: acetylation stabilizes the interaction of the H2A-H2B dimer with the H3-H4 tetramer in nucleosomes that are on highly positively coiled DNA.

A high level of the post-translational modification, acetylation, is found on the N-terminal regions of the core histones H2A, H2B, H3, and H4 and is primarily located in the nucleosomes of active genes. An in vitro transcription system was applied, which utilizes T7 RNA polymerase and template DNAs that are either moderately or highly positively coiled, to determine whether acetylation alters the dynamics of histone displacement from these templates during transcription. To measure displacement, an excess of a competitor (negatively coiled DNA reconstituted with unlabeled H3-H4) was included during the transcription process. Acetylated but not unacetylated (3)H-labeled H3-H4 was found to displace with high frequency from the moderately positively coiled template. This displacement of acetylated H3-H4 was not observed when the template was highly positively coiled. Acetylated (3)H-labeled H2A-H2B also preferentially displaced to the competitor, but in this instance, transcription-induced stress on the highly positively coiled template was required. The histone chaperone, NAP1, was found to facilitate the displacement of both H3-H4 and H2A-H2B. Surprisingly, when acetylated H2A-H2B and acetylated H3-H4 were reconstituted together in the same nucleosomes, the displacement of acetylated H2A-H2B was much reduced during transcription. We conclude that acetylation alters nucleosome stability by enhancing displacement of H3-H4, while decreasing the displacement of H2A-H2B. These results are discussed with regard to potential in vivo conditions in which these observations may be relevant.

Histone release during transcription: displacement of the two H2A-H2B dimers in the nucleosome is dependent on different levels of transcription-induced positive stress.

Both indirect (transcription-induced stress) and direct effects of polymerase elongation on histone-DNA interactions were studied on closed circular DNA that was either moderately or positively coiled. The templates were reconstituted with (3)H-labeled H2A, H2B, H3, and H4 to form nucleosomes, and transcription was done with T7 RNA polymerase in the presence of a negatively coiled competitor DNA (reconstituted with unlabeled H3 and H4). The first of the two labeled H2A-H2B dimers readily displaced from the highly positively coiled template to the competitor even in the absence of transcription, while the indirect effect of transcription-induced stress was required for the moderately coiled template. The second labeled H2A-H2B dimer required transcription-induced stress for both moderately and highly positively coiled DNA. The displacement of the labeled H3-H4 tetramer also occurred, provided it was associated with an H2A-H2B dimer and a moderately positively coiled DNA. This displacement occurred independent of transcription-induced stress and is likely due to the direct effect of polymerase disruption of histone-DNA interactions. The inclusion of the histone chaperone, NAP1, greatly enhanced the release of both of the two H2A-H2B dimers. These observations are consistent with in vivo observations which indicate that during transcription H2A and H2B are significantly more mobile than H3 and H4 and indicate that transcription-induced positive stress is a likely cause for this selective movement.

Histone release during transcription: NAP1 forms a complex with H2A and H2B and facilitates a topologically dependent release of H3 and H4 from the nucleosome.

Transcription through a multinucleosomal template was studied to determine why histones are released to the nascent RNA. It was first determined in competition experiments between DNA and RNA that histones H2A and H2B have a 20-fold preference for binding RNA over DNA; a preference was not seen for histones H3 and H4. Histones H3 and H4 would preferentially bind RNA, provided they were in an octameric complex with H2A and H2B. In transcription studies with T7 RNA polymerase, H3 and H4 were transferred to the nascent RNA, provided the template was linear. If the DNA was topologically restrained, which is a condition that more closely maintains transcription-induced stresses, H3 and H4 would not release. Histones H3 and H4 would be released from this template when H2A and H2B were present, a release that was enhanced by the presence of nucleosome assembly protein-1 (NAP1). Since a small quantity of H2A and H2B is sufficient to facilitate this transfer, it is proposed that H2A and H2B function to repeatedly shuttle H3 and H4 from the template DNA to the RNA. Cross-linked histones (dimethylsuberimidate-cross-linked octamer) were reconstituted into nucleosomes and found to be transferred to the RNA at the same frequency as un-cross-linked histones, an indication that such large complexes can be released during transcription. Transcription was carried out in the presence of Escherichia coli topoisomerase I so that positive coils would accumulate on the DNA. Histones H3 and H4 would again not be transferred from this DNA, unless H2A and H2B were present. In this instance, however, when NAP1 was present, the shuttling of H3 and H4 to the RNA caused a significant depletion of H2A and H2B from the positively coiled DNA. These results are discussed with regard to current models for transcription through nucleosomes.