Closely spaced nucleosome cores in reconstituted histone.DNA complexes and histone-H1-depleted chromatin

It has been demonstrated by digestion studies with micrococcal nuclease that reconstitution of complexes from DNA and a mixture of the four small calf thymus histones H2A, H2B, H3, and H4 leads to subunits closely spaced in a 137 +/- 7-nucleotide-pair register. Subunits isolated from the reconstituted complex contain nearly equimolar amounts of the four histones and sediment at 11.6S. On DNase I digestion both the reconstituted complex and the separated subunits gave rise to series of single-stranded DNA fragments with a 10-nucleotide periodicity. This indicates that the reconstitution leads to subunits very similar to nucleosome cores. Nucleosome cores closely spaced in a 140-nucleotide-pair register were also obtained upon removal of histone H1 from chromatin by dissociation with 0.63 M NaCl and subsequent ultracentrifugation. In reconstitution experiments with all five histones (including histone H1) our procedure did not lead to tandemly arranged nucleosomes containing about 200 nucleotide pairs of DNA. In the presence of EDTA, DNase II cleaved calf thymus nuclei and chromatin at about 200-nucleotide-pair intervals whereas in the presence of Mg2+ cleavage at intervals of approximately half this size was observed. The change in the nature of the cleavage pattern, however, was no longer found after removal of histone H1 from chromatin. This indicates that H1 influences the accessibility of DNase II cleavage sites in chromatin. This finding is discussed with respect to the influence of histone H1 on chromatin superstructure.     

The condensation of chromatin and histone H1-depleted chromatin by spermine

At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

Histone acetylation reduces H1-mediated nucleosome interactions during chromatin assembly

During chromatin replication and nucleosome assembly, newly synthesized histone H4 is acetylated before it is deposited onto DNA, then deacetylated as assembly proceeds. In a previous study (Perry and Annunziato, Nucleic Acids Res. 17, 4275 [1989]) it was shown that when replication occurs in the presence of sodium butyrate (thereby inhibiting histone deacetylation), nascent chromatin fails to mature fully and instead remains preferentially sensitive to DNaseI, more soluble in magnesium, and depleted of histone H1 (relative to mature chromatin). In the following report the relationships between chromatin replication, histone acetylation, and H1-mediated nucleosome aggregation were further investigated. Chromatin was replicated in the presence or absence of sodium butyrate; isolated nucleosomes were stripped of linker histone, reconstituted with H1, and treated to produce Mg(2+)-soluble and Mg(2+)-insoluble chromatin fractions. Following the removal of H1, all solubility differences between chromatin replicated in sodium butyrate for 30 min (bu-chromatin) and control chromatin were lost. Reconstitution with H1 completely restored the preferential Mg(2+)-solubility of bu-chromatin, demonstrating that a reduced capacity for aggregation/condensation is an inherent feature of acetylated nascent nucleosomes; however, titration with excess H1 caused the solubility differences to be lost again. Moreover, when the core histone N-terminal "tails" (the sites of acetylation) were removed by trypsinization prior to reconstitution, H1 was unable to reestablish the altered solubility of chromatin replicated in butyrate. Thus, the core histone "tails," and the acetylation thereof, not only modulate H1-mediated nucleosome interactions in vitro, but also strongly influence the ability of H1 to differentiate between new and old nucleosomes. The data suggest a possible mechanism for the control of H1 deposition and/or chromatin folding during nucleosome assembly.     

Mutual arrangement of histone H1 molecules in extended chromatin. Chymotryptic digestion of cross-linked H1 histone dimers

Mutual arrangement of histone H1 molecules in chromatin extended in low salt-EDTA buffer and additionally in the presence of urea was studied by means of reversible cross-linking combined with chymotryptic digestion. In the chromatins tested, the chymotryptic halves of H1 were cross-linked in all possible combinations; i.e., C-C, C-N and N-N. The results imply that the mutual arrangement of H1 histones is determined by the structure of extended nucleosomal chain, rather than chromatin superstructure.     

Histone crosslinking patterns indicate dynamic binding of histone H1 in chromatin

Crosslinking of histone H1 molecules to each other and to the core histones with bifunctional reagents in mouse liver nuclei and chromatin was compared with that under the conditions of random 'contacts' between these molecules. The patterns of crosslinking of the H1 subfractions (H1A, H1B, and H10) to each other in nuclei, chromatin and in solution at different ionic strengths due to random collisions were essentially the same. Moreover, the contacts between the H1 molecules were qualitatively the same in nuclei, chromatin and in solution also at the level of the chymotryptic halves of the H1 molecules. The contacts between the H1 molecules and the core histones in nuclei were similar to those obtained in chromatin at 70 mM NaCl, when H1 molecules readily migrate, and at 0.6 M NaCl, when H1 molecules are dissociated from chromatin. We conclude that spatial arrangement of H1 subfractions and mutual orientation of H1 molecules in isolated nuclei are random-like at least in terms of cross-linking. The static and dynamic models of histone H1 binding to chromatin compatible with the known data are considered. Although unequivocal verification of the models is not possible at present, the dynamic models do correspond better to recent data on the location of the histone H1 in nuclei and chromatin.     

Histone H1 and chromatin higher order structure. Does histone H1 exhibit specific self-association?

1. Histones H1 and H5 in chromatin and in free solution can be cross-linked to higher multimers. Is this due to a specific protein/protein interaction? If so, this interaction might be the structural basis of the condensation of the chromosomal nucleofilament, known to be mediated by histones H1 and H5. 2. Since only the central domain of H1 and H5 exhibits tertiary folding and globular structure, this is the most likely site of specific interaction. 3. Formaldehyde has been used to test whether the central domains of histone H1 from calf thymus or from sea urchin sperm or histone H5 from chicken erythrocytes self-interact. 4. The cross-linking shown by each globular peptide was compared with that of its parent histone. 5. In all three cases the peptide cross-linked to a much lower extent than its intact parent histone and the observed cross-linked rates were roughly in proportion to the relative number of lysine residues parent histone and peptide. 6. It is concluded that there is no specific self-interaction between the globular domains of either H1 or H5 molecules in free solution. 7. This result suggests that specific H1/H1 protein/protein interactions are not the basic cause of chromatin condensation.     

Atomic force microscopy sees nucleosome positioning and histone H1-induced compaction in reconstituted chromatin.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.     

Free DNA stretches in histone H1-depleted chromatin and their possible relation to chromomere structure

Oligomers of chromatin subunits (oligonucleosomes) were prepared by a mild digestion of chromatin with staphylococcal nuclease followed by a purification of a high molecular weight material (hexanucleosomes and larger DNP particles) by gel chromatography. The main finding is that a mild removal of histone H1 from the oligonucleosome preparation by treatment with tRNA in the absence of any significant hydrodynamic shearing leads to the formation of free DNA molecules which constitute 5–6% of the total oligonucleosomal DNA.The size of nucleosome-free DNA stretches in H1-depleted hydrodynamically sheared chromatin is about 6000 base pairs and their content is apparently 10–12% of the total DNA. These and related findings are discussed in terms of the previously proposed asymmetric hairpin model of DNA packing in chromatin [1–4]. Different kinds of the asymmetric hairpin are considered and ambiguities in interpretations of experimental data are pointed out.     

Nuclease digestion promotes structural rearrangements in H1-depleted chromatin.

Digestion of H1-depleted chromatin with micrococcal nuclease at an ionic strength of 0.35M gives rise to structural rearrangements indicating nucleosomal sliding. The ionic strength necessary to reveal this effect is significantly lower than that required in the absence of an accompanying digestion. As an explanation, a model is presented in which the progressing terminal degradation of oligomeric nucleosomes is made responsible for promoting structural rearrangements.     

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.     

The structure and dynamics of H1-depleted chromatin

The size of DNA involved in the interaction with a histone octamer in H1-depleted chromatin was re-examined. We compared the thermal untwisting of chromatin DNA and naked DNA using CD and electrophoretic topoisomer analysis, and found that DNA of 175 +/- 10 base pairs (bp) in length interacted with the histone core under physiological conditions. The decrease of ionic strength below 20 mM NaCl reduced this length down to 145 bp: apparently, an extra 30 bp DNA dissociated from the histone core to yield well-known 145-bp core particle. Histone cores partly dissociate within the temperature range of 25 to 40 degrees C. Quantitative analysis of histone thermal dissociation from DNA shows that the size of DNA protected against thermal untwisting would be significantly overestimated if this effect is neglected. The results presented in this paper also suggest that the dimers (H2A, H2B) act as a lock, which prevents transmission of conformational alterations from a linker to nucleosome core DNA. The histone core dissociation as well as (H2A, H2B) dimer displacement are discussed in the light of their possible participation in the eukaryotic genome activation.     

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.