Preferential and asymmetric interaction of linker histones with 5S DNA in the nucleosome.

We establish that linker histones H1 and H5 bind preferentially to a Xenopus borealis somatic 5S RNA gene associated with an octamer of core histones rather than to naked 5S DNA. This preferential binding requires free linker DNA to either side of the nucleosome core. Incorporation of a single linker histone molecule into the nucleosome protects an additional 20 bp of linker DNA from micrococcal nuclease digestion. This additional DNA is asymmetrically distributed with respect to the nucleosome core. Incorporation of linker histones causes no change to the cleavage of DNA in the nucleosome by hydroxyl radical or DNase I.     

Histone contributions to the structure of DNA in the nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.     

Nucleosome positioning is determined by the (H3-H4)2 tetramer.

It is demonstrated that the histone (H3-H4)2 tetramer can find specific positions on DNA, even in the absence of other histones. Purified histone (H3-H4)2 tetramers were reconstituted onto 208-base-pair (bp) DNA molecules containing a nucleosome-positioning sequence by using salt-gradient dialysis. The stoichiometry of histone tetramer to DNA was shown to be 1:1. Digestion with micrococcal nuclease led to formation of protected DNA fragments of approximately 73 bp. Cleavage of the 73-bp DNA with restriction enzymes produced a small set of defined bands, demonstrating positioning of the (H3-H4)2 tetramer on DNA. Analysis of the restriction digests shows that the 73-bp DNA corresponds mainly to two fragments, one lying on either side of the pseudo-dyad axis of the major position adopted by complete histone octamers on this DNA. This result means that a single (H3-H4)2 histone tetramer can fold approximately 146 bp of DNA with the same positioning as the complete octamer but that a region near the pseudo-dyad is only weakly protected against micrococcal nuclease attack in the absence of histones H2A and H2B.     

Contacts of the globular domain of histone H5 and core histones with DNA in a "chromatosome".

The globular domain of histone H5 is found to asymmetrically associate with a nucleosome core including the Xenopus borealis somatic 5S RNA gene. Histones H2A and H2B are required for association of histone H5. Strong crosslinking of the globular domain of histone H5 to the 5S DNA in the nucleosome occurs at a single site to one side of the dyad axis. This site is also in contact with the core histones, and the interactions of the core histones with 5S DNA change as a result of association of the globular domain of histone H5. We discuss evidence for an allosteric change in core histone-5S DNA interactions following the association of the linker histone in the nucleosome.     

In vitro core particle and nucleosome assembly at physiological ionic strength.

Nucleosome core particles have been efficiently assembled in vitro by direct interaction of histones and DNA at physiological ionic strength, as assayed by digestion with DNases, supercoiling of relaxed circular DNA, and electron microscopy. Reconstitution was achieved either by the simultaneous addition of all core histones, or by the sequential binding of H3 . H4 tetramer and H2A . H2B dimer to DNA. Micrococcal nuclease digestion and electron microscopy studies indicated that there is heterogeneity in the spacings at which core particles are assembly on the DNA. Length measurements of oligomeric DNA produced during the course of the digestion suggest that the core histone octamer can organize 167 (+/- 4) rather than 145 base pairs of DNA, the extra 20 base pairs being quickly digested. Binding of histone H1 to core particles resulted in the protection of about 165 base pairs of DNA from nuclease attack. Because the core histone octamer is fully dissociated into H3 . H4 tetramer and H2A . H2B dimer at physiological ionic strength, our results would suggest that in vivo core particle assembly may also occur by interaction of these two complexes on the nascent DNA.     

Composition of native and reconstituted chromatin particles: direct mass determination by scanning transmission electron microscopy.

Chromatin particles reconstituted from 145-base-pair lengths of DNA and either the arginine-rich histones H3 and H4 only or all four nucleosomal core histones have been compared with native nucleosomes in terms of their ultrastructure and mass distribution, as determined by scanning transmission electron microscopy (STEM). The mass of the nucleosome derived from STEM analysis was very close to that calculated for its DNA and histone components. The reconstituted particles showed a broader mass distribution, but it was clear that the majority contained at least eight histone molecules. This was to be expected for structures reconstituted from all four core histones, but in the case of H3H4-DNA complexes clearly showed that an octamer rather than tetramer of these histones was required to fold nucleosomal DNA into a stable compact particle. The significance of the H3H4 octamer complex with respect to nucleosomal structure is discussed, and the evidence that nucleosomal DNA can accept even greater numbers of histones is considered.     

Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles: Potential flipping of the protein from a left- to a right-handed superhelical form.

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two H3s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a left- to a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. & Moudrianakis, E. N. (1991) Proc. Natl.Acad. Sci. USA 88, 10148-10152] that this change in DNA topology is mediated by a similar change in the topology of the tetramer itself, which may occur through a rotation (or a localized deformation) of the two H3-H4 dimers about their H3-H3 interface. Potential implications of this model for nucleosome dynamics in vivo are discussed.     

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.     

Rearrangement of the histone H2A C-terminal domain in the nucleosome.

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.     

The tail domain of lamin Dm0 binds histones H2A and H2B

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.     

Anti-chromatin (anti-nucleosome) antibodies

Chromatin is the native complex of histones and DNA found in the cell nucleus of eukaryotes. The fundamental subunit of chromatin is the nucleosome, which is composed of a core particle in which 146 bp of helical DNA are wrapped around an octamer made up of two H2A-H2B dimers that surround an H3-H4 tetramer. The prevalence of anti-chromatin (nucleosome) antibodies in systemic lupus erythematosus (SLE) varies from 50% to 90%, being similar to that of the classical positive LE cell. The presence of these antibodies can be used, in conjunction with clinical findings and other laboratory tests, to help in the diagnosis of SLE and drug induced lupus. The presence of anti-chromatin antibodies has also been linked to glomerulonephritis in SLE patients.     

Histone H3 disulfide dimers and nucleosome structure.

The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and H4. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations. The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.     

Histone packing in the nucleosome core particle of chromatin

The chromatin core particle DNA conformation deduced in broad outline by Finch et al. [Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B., Levitt, M. & Klug, A. (1977) Nature 269, 29-36] can be described in detail using other available experimental results. Histone binding sites compatible with the pattern of pancreatic DNase I digestion (Simpson, R. T. & Whitlock, J. P., Jr. (1976) Cell 9, 347-353; Noll, M. (1977) J. Mol. Biol. 116, 49-71; Lutter, L. C. (1977) J. Mol. Biol. 117, 53-69] lend to core particle DNA pseudosymmetry characteristic of molecular point group D(3). DNA symmetry and pseudosymmetry, in turn, imply equivalence and quasi-equivalence properties of the histone packing arrangement that support the following deductions: (i) One and only one alpha(2)beta(2) histone tetramer, presumably (H3)(2)(H4)(2), can serve as a stable subassembly within the histone octamer. (ii) There is a unique, strand-specific way to assign DNA binding domains to the arginine-rich histones (H3 and H4). (iii) Histones H3 and H4 alone should suffice to impose a supercoiled structure on DNA, as is observed experimentally, because only the tetramer can mimic a screw dislocation and thereby complement the screw symmetry of the DNA supercoil. (iv) The two slightly lysine-rich histones H2A and H2B are probably responsible, each in a different way, for dividing the eukaryotic chromatin fiber into discrete subunits. (v) The proposed arrangement of four distinct proteins appears to be a minimum formal requirement for making nucleosomes; that is, for introducing regularly spaced supercoiled DNA folds without also allowing formation of an indefinitely long (and genetically inert) DNA superhelix.     

Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro

Centromere protein A (CENP-A) is a variant of histone H3 with more than 60% sequence identity at the C-terminal histone fold domain. CENP-A specifically locates to active centromeres of animal chromosomes and therefore is believed to be a component of the specialized centromeric nucleosomes on which the kinetochores are assembled. Here we report that CENP-A, highly purified from HeLa cells, can indeed replace histone H3 in a nucleosome reconstitution system mediated by nucleosome assembly protein-1 (NAP-1). The structure of the nucleosomes reconstituted with recombinant CENP-A, histones H2A, H2B, and H4, and closed circular DNAs had the following properties. By atomic force microscopy, "beads on a string" images were obtained that were similar to those obtained with nucleosomes reconstituted with four standard histones. DNA ladders with repeats of approximately 10 bp were produced by DNase I digestion, indicating that the DNA was wrapped round the protein complex. Mononucleosomes isolated by glycerol gradient sedimentation had a relative molecular mass of approximately 200 kDa and were composed of 120-150 bp of DNA and equimolar amounts of CENP-A, and histones H4, H2A, and H2B. Thus, we conclude that CENP-A forms an octameric complex with histones H4, H2A, and H2B in the presence of DNA.