Histones and nucleosomes in Archaea and Eukarya: a comparative analysis

Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3+H4)₂ tetramer. Received: January 22, 1998 / Accepted: February 16, 1998     

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.     

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.     

H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro

Nucleosome formation has been studied in a system containing relaxed Col E1 DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assembled in the presence of the H1 histone, polylysine (Mr 20 000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.     

Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.

High speed supernatants of Xenopus laevis oocyte nuclei efficiently assemble DNA into nucleosomes in vitro under physiological salt conditions. The assembly activity cofractionates with two histone complexes composed of the acidic protein N1/N2 in complex with histones H3 and H4, and nucleoplasmin in complex with histones H2B and H2A. Both histone complexes have been purified and their nucleosome assembly activities have been analysed separately and in combination. While the histones from the N1/N2 complexes are efficiently transferred to DNA and induce supercoils into relaxed circular plasmid DNA, the nucleoplasmin complexes show no supercoil induction, but can also transfer their histones to DNA. In combination, the complexes act synergistically in supercoil induction thereby increasing the velocity and the number of supercoils induced. Electron microscopic analysis of the reaction products shows fully packaged nucleoprotein structures with the typical nucleosomal appearance resulting in a compaction ratio of 2.8 under low ionic strength conditions. The high mobility group protein HMG-1, which is also present in the soluble nuclear homogenate from X. laevis oocytes, is not required for nucleosome core assembly. Fractionation experiments show that the synergistic effect in the supercoiling reaction can be exerted by histones H3 and H4 bound to DNA and the nucleoplasmin complexes alone. This indicates that it is not the synchronous action of both complexes which is required for nucleosome assembly, but that their cooperative action can be resolved into two steps: deposition of H3 and H4 from the N1/N2 complexes onto the DNA and completion of nucleosome core formation by addition of H2B and H2A from the nucleoplasmin complexes.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.     

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=     

Nucleosome-like Complex of the Histone from the Hyperthermophile Methanopyrus Kandleri (MkaH) with Linear DNA

Abstract

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (Rw) of ≈3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw ≤ 3 revealed formation of isometric loops encompassing 71 +/- 7 bp of DNA duplex. At high values of Rw (≥9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)₂ tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.     

DNA wrapping in nucleosomes. The linking number problem re-examined.

Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure.     

Nucleosome-like complex of the histone from the hyperthermophile Methanopyrus kandleri (MkaH) with linear DNA

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (R(w)) of approximately 3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw or=9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)(2) tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.     

Archaeal histone selection of nucleosome positioning sequences and the procaryotic origin of histone-dependent genome evolution

Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages.     

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.     

The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers.

BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.     

MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension

All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three α-helices separated by two β-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647Δ) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)₄ tetramer, were apparently less stable than those formed by (rMJ1647Δ)₄. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647Δ in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones. Received: September 2, 1999 / Accepted: October 18, 1999     

NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This method allows to apply a well-defined stretching force and supercoiling density to a single DNA molecule, and to study in real time the change in linking number, stiffness and length of the DNA during nucleosome formation. We observe a decrease in end-to-end length when NAP1 and core histones (CH) are added to the dsDNA. We characterize the formation of complete nucleosomes by measuring the change in linking number of DNA, which is induced by the NAP1-assisted nucleosome assembly, and which does not occur for non-nucleosomal bound histones H3 and H4. By rotating the magnets, the supercoils formed upon nucleosome assembly are removed and the number of assembled nucleosomes can be counted. We find that the compaction of DNA at low force is about 56 nm per assembled nucleosome. The number of compaction steps and associated change in linking number indicate that NAP1-assisted nucleosome assembly is a two-step process.