The superstructure of chromatin and its condensation mechanism

Synchroton radiation X-ray scattering experiments have been performed on chicken erythrocyte chromatin fibres over a wide range of ionic conditions and on various states of the fibres (i.e. "native" in solution, in gels and in whole nuclei; chromatin depleted of the H1 (H5) histones and chromatin with bound ethidium bromide). A correlation between the results obtained with the various chromatin preparations provides evidence for a model according to which at low ionic strength the chromatin fibre already possesses a helical superstructure, with a diameter comparable to that of condensed chromatin, held together by the H1(H5) histone. The most significant structural modification undergone upon an increase of the ionic strength is a reduction of the helix pitch, this leads to condensation in a manner similar to the folding of an accordion. The details of this process depend on whether monovalent or divalent cations are used to raise the ionic strength, the latter producing a much higher degree of condensation. Measurements of the relative increase of the mass per unit length indicate that the most condensed state is a helical structure with a pitch around 3.0-4.0 nm. In this paper we give a detailed presentation of the experimental evidence obtained from static and time-resolved scattering experiments, which led to this model.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.     

Chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength and contain linker histones are highly enriched in beta-globin gene sequences.

Mature chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength are enriched in beta-globin DNA sequences. Vitellogenin chromatin, which is not expressed in this tissue, is found in aggregation prone, salt insoluble chromatin. There is a direct correlation between the size of soluble fragments and the degree of globin gene enrichment, with the largest fragments being most highly enriched. The highly globin enriched (about 50 fold) polynucleosomes contain significantly elevated levels of acetylated histones H4, H2A.Z, and H2B, and ubiquitinated (prefix "u") histones H2A and H2B (with a significant relative increase of uH2B over uH2A). These polynucleosomes were complexed with histones H1 and H5 but at a lower level than that found in unfractionated chromatin.     

Comparison of the primary structure of reconstructed minimal nucleosomes and nucleosomes isolated from the chromatin

We have reconstructed nucleosomes from a histone octamer (H2A, H2B, H3, H4)2 and DNA 146 b.p. or 2-3 thousands b.p. in length. Comparison by means of DNA-histone cross-links of the primary organization of minimal nucleosomes obtained by reconstruction or isolated from chromatin of chicken erythrocyte nuclei has demonstrated a high similarity in histone location on their DNAs. Simultaneously, there have been observed some variations in the character of interaction for all core histones with DNA on nucleosomes. Thus, the cross-link of histone H4 with DNA of a core particle at H4 sites (65), unlike H4(55) and H4(88) sites, significantly depends on the superstructure of chromatin, ionic strength of solution and the presence of denaturating agents. All these differences are expected to probe the existence of conformational isomers for core particles. (Bracketed is the distance from the histone interaction site with the DNA of the core particle to the DNA 5'-terminus.)     

Effect of the supranucleosomal chromatin organization on histone-DNA interrelations

It has been demonstrated by the method of competitive displacement of own chromatin histone by excess total histone that chromatin dispersity influence the strength of histone-DNA interactions in a medium of physiological ionic strength. Histone NI was removed from chromatin after the quantity of total histone added to chromatin was equivalent to that existing in chromatin. The proportion of histones H2A and H2B removed from chromatin was increased after mechanical of ultrasonic degradation of chromatin at 5-20-fold excess of total extra-histone. In some histone preparations, the removal of histones H2A and H2B was not detectable at even 200-fold excess of total histone. This may be explained by strengthening histone-DNA interactions in superhelical loops of chromatin.     

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.     

Viscosity of chromatin solutions increases with increasing ionic strength

Increasing the ionic strength of rat liver chromatin solutions above 0.4 M causes increasing viscosity. This indicates transformation of the compact chromatin molecules to more elongated forms. In the range of 0.4–0.5 M ionic strength histone H1 is dissociating continuously from the chromatin and the quaternary structure chromatin unravels. At ionic strength higher than 0.5 M the viscosities of chromatin solutions are furthermore increasing due to structural deformation. Near 0.7 M ionic strength the core histones H2A and H2B begin to dissociate from the chromatin, and the opening of the nucleosome cores leads to increasing elongation of the chromatin molecules.     

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.     

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

Effects of histone acetylation on nucleosome properties as evaluated by polyacrylamide gel electrophoresis and hydroxylapatite dissociation chromatography

The release of acetylated histones from chick oviduct chromatin was analyzed by hydroxylapatite column chromatography. By raising of the NaCl concentration, acetylated histones were eluted from hydroxylapatite-bound chromatin depending on their release from nucleosomal DNA. Electrophoresis on acid-urea gel showed that hyperacetylated forms of histone H4 were eluted at a lower NaCl concentration than non-acetylated or hypoacetylated H4, suggesting that hyperacetylated H4 has decreased stability in nucleosomes. However, under milder ionic conditions which do not induce dissociation between histones and DNA, polyacrylamide gel electrophoresis of purified nucleosome cores showed no evidence for their unfolding or for increased accessibility by high mobility group protein-17.     

Location of the ethidium binding sites of high affinity in chromatin

The influence of H1 and H5 histones proteins upon the accessibility of ethidium bromide into chromatin is studied by steady-state fluorescence anisotropy in the range of r-values ([Dye]/[Phosphate]) smaller than 0.01. This corresponds to the very strong binding process. When H1 and H5 are present, the DNA segment which contains the binding sites is 25–30 base pairs long, even if H1 and H5 are digested by trypsin or by natural proteolysis, but presumably still interacting with the DNA chromatin. On the contrary, when H1 or H5 are separated from chromatin by an increase of the ionic strength, ethidium binds to a segment of DNA about 55–60 base pairs long. We may explain the results by assuming that the ethidium sites are located on a continuous segment constituting about one half of the linker, the other half interacting with H1 and H5. When chromatin is depleted from these proteins, the high affinity sites are distributed all along the linker.     

Relaxation of chromatin structure upon removal of histones H2A and H2B

Modification of chromatin from chicken erythrocytes with dimethylmaleic anhydride is accompanied by its solubilization and the dissociation of histones H1, H5, H2A and H2B. Histone H1 is the first to dissociate and H5 the last. After regeneration of the modified amino groups, residual chromatin preparations with different histone composition were studied by circular dichroism and thermal denaturation. In addition to the effects produced by the lack of histones H1 and H5, both techniques show a substantial relaxation of chromatin structure induced by the loss of histones H2A and H2B, which appear to play an important role in the superhelical folding of DNA.     

Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications.

The modification patterns of histones present in various forms of intracellular simian virus 40 nucleoprotein complexes were analyzed by acetic acid-urea-polyacrylamide gel electrophoresis. The results showed that different viral nucleoprotein complexes contain different histone patterns. Simian virus 40 chromatin, which contains the activities for the synthesis of viral RNA and DNA, exhibits a histone modification pattern similar to that of the host chromatin. However, virion assembly intermediates and mature virions contain highly modified histones. Pulse-chase experiments with [3H]lysine showed that the newly incorporated histones in the virion assembly intermediates were already highly modified. The majority of in vivo acetylation activity of histones occurred on the 70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate. These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into the mature virion: (i) modification of histones, (ii) accumulation of capsid protein around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion assembly is discussed.     

Histone H1 can be removed selectively from chicken erythrocyte chromatin at near physiological conditions.

Histone H1 was depleted selectively from chicken erythrocyte polynucleosomes, without any detectable concomitant loss of H5 or core particle histones. The depletion is performed with ion exchange resin at low ionic strength (80 mM NaCl). The nucleosomes did not slide during the procedure. In contrast to the native chromatin, H1 depleted polynucleosomes are completely soluble in the 5--600 mM NaCl range.     

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.     

Structural transition in chromatin induced by ions in solution

Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones.