Linker histone-dependent DNA structure in linear mononucleosomes

We have examined the binding of the linker histone H5 (LH) to mononucleosomes. Mononucleosomes reconstituted on short DNA fragments display a series of discrete bands on a gel corresponding to various nucleosome positions along the DNA. When a series of engineered H5s with differing extents of the C-terminal tail are bound to these mononucleosomes, the electrophoretic mobilities of the resulting complexes are altered. Not only is there a general increase in mobility upon complex formation, but there is a reduction in the differences in mobility of the most distal nucleosomes. The complexes were also visualized by electronmicroscopy. From these two complementary studies, we conclude the following. (1) Entering and exiting DNAs are uncrossed in the LH-free particles, despite a DNA wrapping of 1.65 to 1.7 turns around the histone core. This results from a bending of the entering and exiting DNA away from each other and the histone surface, presumably as a consequence of electrostatic repulsion. This confirms and extends conclusions derived from our recent examination of the same particles in 3D through cryo-electron microscopy. (2) Binding of the globular domain of H5 increases DNA wrapping to 1.8 to 1.9 turns, but fails to induce a crossing due to an accentuation of the bends. (3) The C-terminal tail of H5 bridges entering and exiting DNAs together into a four-stranded stem over a distance of about 30 bp. The occurrence of such a stem may introduce constraints on models of the 30 nm chromatin fiber.     

Archaeal nucleosomes

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.     

Nucleosome positioning on chicken and human globin gene promoters in vitro. Novel mapping techniques

We have developed two new techniques to assess the positions adopted by core histone octamers when reconstituted onto DNA. These, together with a previously described technique, were applied to mapping binding sites on plasmid DNAs containing either the human zeta-globin or chicken beta-globin gene promoters. Each of the approaches enabled the sites occupied by histone octamers to be measured at high resolution and, in qualitative terms, revealed the same pattern of multiple, overlapping sites. Monomer extension, one of the novel techniques, can be used to reveal binding sites over extensive stretches of a single reconstitute (approximately 1000 bp). We found the distribution of histone octamer binding sites to be largely independent of the conditions employed for reconstitution, the topology of the DNA substrate and prolonged incubation under various post-reconstitution conditions. These properties, and features of the binding site maps that we derived, suggest that histone octamer positioning on these DNAs is predominantly a characteristic of the DNA sequence itself and, by implication, that nucleosome-nucleosome interactions and the formation of nucleosome arrays are of minor influence. Some of the techniques provide quantitative information concerning the relative binding strengths of the core histone octamer for different positioning sequences. In this context, it is notable that the majority of potential binding sites compete very poorly for the histone octamer, demonstrating that under the conditions pertinent to our analysis, the range of binding strengths exhibited by the octamer for particular DNA sequences is extensive, and greater than that observed when competitive binding has been studied by methods that do not reflect precise positioning.     

A topological approach to nucleosome structure and dynamics: the linking number paradox and other issues

The linking number paradox of DNA in chromatin (two negative crossings around the octamer, associated with a unit linking number reduction), which is 21 years old this year, has come of age. After stirring much debate in the past, the initially hypothetical explanation of the paradox by DNA overtwisting on the nucleosome surface is now presented as a hard fact in recent textbooks. The first part of this article presents a historical perspective of the problem and details the numerous attempts to measure DNA local periodicity, which in one remarkable example sowed the seeds for the discovery of DNA bending. The second part is devoted to the DNA minicircle system, which has been developed in the author's laboratory as an alternative to the local-periodicity-measurement approach. It offers a simple proposal: a unit linking number reduction associated with a single crossing. This conclusion is contrasted with the latest high-resolution crystallographic data of the nucleosome in the third part of the article, and the fourth part examines the available evidence supporting an extension of these results to nucleosomes in chromatin. The last part addresses another basic question pertaining to nucleosome dynamics, the conformational flexibility of the histone tetramer.     

The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel

We have examined the role played by various histones in the organization of the DNA of the nucleosome, using staphylococcal nuclease as a probe of DNA conformation. When this enzyme attacks chromatin, a series of fragments evenly spaced at 10 base pair intervals is generated, reflecting the histone-DNA interactions within the nucleosome structure. To determine what contribution the various histones make to DNA organization, we have studied the staphylococcal nuclease digestion patterns of complexes of DNA with purified histones. Virtually all possible combinations of homogeneous histones were reconstituted onto DNA. Exhaustive digestion of a complex containing the four histones H2A, H2B,H3, and H4 yields a DNA fragment pattern very similar to that of whole chromatin. The only other combinations of histones capable of inducing chromatin-like DNA organization are H2A/H2B/H4 and those mixtures containing both H3 and H4. From an examination of the kinetics of digestion of H3/H4 reconstitutes, we conclude that although the other histones have a role in DNA organization within the nucleosome, the arginine-rich histone pair, H3/H4, can organize DNA segments the length of the nucleosome core in the absence of all other histones.     

Linker DNA and H1-dependent reorganization of histone-DNA interactions within the nucleosome

We have employed a site-directed photochemical cross-linking procedure to precisely map interactions between nucleosomal DNA and the C-terminal tail of core histone H2A. We find that this tail has the potential to contact multiple sites within the nucleosome and that these contacts are dependent upon the configuration of the complex. This tail contacts DNA near the dyad axis within nucleosome core particles but rearranges to a site near the edge of the nucleosomal DNA when linker DNA is present. Moreover, in the presence of linker histone H1 the contacts near the edge of the nucleosome but not at the dyad are further rearranged. In addition, we present further evidence for the suggestion that the binding of linker histone causes a subtle but global change in core histone-DNA interactions within the nucleosome [Usachenko, S. I., Gavin, I. M., and Bavykin, S. G. (1996) J. Biol. Chem. 271, 3831-3836].     

Nucleosome packaging and nucleosome positioning of genomic DNA

The goals of this study were to assess the extent to which bulk genomic DNA sequences contribute to their own packaging in nucleosomes and to reveal the relationship between nucleosome packaging and positioning. Using a competitive nucleosome reconstitution assay, we found that at least 95% of bulk DNA sequences have an affinity for histone octamer in nucleosomes that is similar to that of randomly synthesized DNA; they contribute little to their own packaging at the level of individual nucleosomes. An equation was developed that relates the measured free energy to the fractional occupancy of specific nucleosome positions. Evidently, the bulk of eukaryotic genomic DNA is also not evolved or constrained for significant sequence-directed nucleosome positioning at the level of individual nucleosomes. Implications for gene regulation in vivo are discussed.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.     

Modeling protein-induced configurational changes in DNA minicircles

A method is offered for obtaining minimum energy configurations of DNA minicircles constrained by one or more DNA-binding proteins. The minicircles are modeled as elastic rods, while the presence of bound protein is implied by rigidly fixing portions of these chains. The configurations of the geometrically constrained circular rods are sampled stochastically and optimized according to a simple elastic energy model of nicked DNA. The shapes of the minimum energy structures identified after a simulated annealing process are analyzed in terms of relative protein orientation and writhing number. The procedure is applied to minicircles 500 base pairs in length, bound to two evenly spaced DNA-wrapping proteins. The presence of histone octamers is suggested by rigidly fixing the two protein-bound portions of each minicircle as small superhelices similar in dimension to nucleosomal DNA. The folded minimum energy forms of sample chains with different degrees of protein wrapping are noteworthy in themselves in that they offer a new resolution to the well-known minichromosome linking number paradox and point to future minicircle simulations of possible import.     

Nucleosome assembly on telomeric sequences

The organization of telomeric chromatin differs from that of bulk chromatin in some peculiar features, such as the unusually short nucleosomal spacing found in vertebrates. Telomeric DNAs are straight, since they consist mostly of 6-8-bp repeated sequences, therefore out of phase with the B DNA period. This feature should be of relevance in nucleosome formation, suggesting the usefulness of studying simple model systems of nucleosome assembly. We reconstituted nucleosomes in vitro, by using purified histone octamers and/or by octamer transfer from chicken erythrocyte nucleosomes, onto telomeric sequences from human, Arabidopsis thaliana, and Saccharomyces cerevisiae. All of these telomeres contain GGG and GGT triplets but are characterized by different repeat lengths (6, 7, and 8 bp). The free energies involved in the association process are the highest among the biological sequences so far assayed, suggesting a main role of DNA flexibility in the assembly of telomeric chromatin. Digestion studies with DNase I, hydroxyl radicals, exonuclease III, and lambda exonuclease indicate that telomeric nucleosomes are characterized by multiple translational positioning without rotational phasing, whereas the telomeric DNA folding around the histone octamer shows the canonical periodicity of about 10.2 bp. The experimental results and a theoretical simulation of DNase I digestion indicate a multiple nucleosome positioning with the periodicity of telomeric DNA. This suggests a main role of local chemical recognition between telomeric sequences and the histone octamer in nucleosome assembly.     

Interaction of mithramycin with DNA fragments complexed with nucleosome core particles: comparison with distamycin and echinomycin

We have studied the sequence-specific interaction of mithramycin with nucleosome core particles which have been reconstituted with various DNA fragments. Mithramycin binds to these DNAs without disrupting the integrity of the nucleosome and produces clear DNase I footprints centered around GC-rich regions. In some instances, the footprints produced on free DNA are resolved into two or more smaller sites when the DNA is complexed with the nucleosome core. In a few cases, novel footprints are produced in sequences which did not bind the drug in free DNA samples. The results are explained by suggesting mithramycin binds to GC-rich regions in which the minor groove faces away from the protein core, and which possess a wider than normal narrow groove on account of their location. Hydroxyl radical footprinting and diethyl pyrocarbonate modification confirm that mithramycin does not affect the rotational positioning of the nucleosome-bound DNA. Although distamycin and echinomycin induce novel DNase I digestion products in nucleosomal DNA which are consistent with the proposed change in DNA positioning [Low, C. M. L., Drew, H. R., & Waring, M. J. (1986) Nucleic Acids Res. 14, 6785-6801], hydroxyl radical footprinting and diethyl pyrocarbonate modification suggest these ligands do not change the rotational positioning of the DNA on the nucleosome cores.     

Concomitants of short-and long-term calcitonin therapy

When Acetabularia cliftonii chloroplast DNA (p = 1.706 g/cm3) is centrifuged in an ethidium bromide-CsCl gradient, the lower band is enriched for DNA with a buoyant density of 1.712 g/cm3 containing small covalently closed circular molecules. The minicircles measure 4.15 +/- 0.30 mum in the closed conformation and 4.35 +/- 0.20 mum in the open conformation. They are not of nuclear or bacterial origin, and appear to exist as independent entities within the chloroplast, although a mitochondrial origin cannot be completely ruled out. No 40-45 mum circles, as found in other chloroplasts, were found in either ethidium bromide-CsCl fraction. None were found in total chloroplast DNA by any of a number of methods tried. Linear molecules up to 200 mum were measured in chloroplast lysates. The main chloroplast genome may consist of very large circular molecules which are broken by even small shear forces.     

A signal encoded in vertebrate DNA that influences nucleosome positioning and alignment

Evidence is provided that the nucleotide triplet con-sensus non-T(A/T)G (abbreviated to VWG) influences nucleosome positioning and nucleosome alignment into regular arrays. This triplet consensus has been recently found to exhibit a fairly strong 10 bp periodicity in human DNA, implicating it in anisotropic DNA bendability. It is demonstrated that the experimentally determined preferences for nucleosome positioning in native SV40 chromatin can, to a large extent, be pre-dicted simply by counting the occurrences of the period-10 VWG consensus. Nucleosomes tend to form in regions of the SV40 genome that contain high counts of period-10 VWG and/or avoid regions with low counts. In contrast, periodic occurrences of the dinucleotides AA/TT, implicated in the rotational positioning of DNA in nucleosomes, did not correlate with the preferred nucleosome locations in SV40 chromatin. Periodic occurrences of AA did correlate with preferred nucleosome locations in a region of SV40 DNA where VWG occurrences are low. Regular oscillations in period-10 VWG counts with a dinucleosome period were found in vertebrate DNA regions that aligned nucleosomes into regular arrays in vitro in the presence of linker histone. Escherichia coli and plasmid DNA, which fail to align nucleosomes in vitro, lacked these regular VWG oscillations.