Histone redistribution and conformational effect on chromatin induced by formaldehyde

Histone redistributions between endogenous DNA in calf thymus chromatin and exogenous DNA from Clostridium perfringens (69% A + T) or from Micrococcus luteus (30% A + T) induced by 0.6 M NaCl or by 2% formaldehyde were studied by thermal denaturation. The observed redistribution occurred on histone Hl when the exogenous DNA was (A + T)-richer than the DNA in chromatin, and when the mixture was exposed to 0.6 M NaCl or formaldehyde. When a (G + C)-richer DNA was added as the acceptor for histones, no substantial transfer of histones from chromatin DNA to exogenous DNA was found. Thus the activation energy of histone dissociation from chromatin DNA seems to be substantially lowered by 0.6 M NaCl or formaldehyde such that histones (mostly histone Hl) can be dissociated and bind the (A + T)-richer DNA and form a more stable complex. It is suggested that the formaldehyde effect on histones may be due to the loss of positive charges on lysine and arginin residues (probably more on lysine than on arginine) in histones after their rapid reaction with formaldehyde. Formaldehyde treatment of chromatin also distorts the DNA conformation, as revealed by circular dichroism (CD) studies. This structural effect occurs mainly on those base pairs bound by histones other than Hl, or within the chromatin subunit. Histone redistribution is treated as a thermodynamic phenomenon of histone binding to DNA. The validity of using formaldehyde to study chromatin structure is discussed.     

Histone synthesis in Trypanosoma cruzi

Trypanosoma cruzi is an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence of Trypanosoma histones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence of T. cruzi histones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.     

Physicochemical properties of calcium phosphate coatings produced by pulsed laser deposition at different water vapour pressures

Conformational peculiarities of DNA complexes with histones of the H1 family have been studied by the method of circular dichroism (CD). The H1 histones were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius, starfish Aphelasterias japonica, and bivalve mollusc Chlamis islandicus and also from the rat thymus. It is shown that these sperm-specific histones do not compact DNA in low ionic strength solution. At physiologic conditions H1 from the sea urchin and starfish sperms compact DNA more intensively than other histones. The H1 from rat thymus has a minimum ability to compact DNA. This histone does not change the structure of DNA double helix. It was supposed that it could be associated with interactions of this histone with DNA in the major groove of its helix. At the same time sperm-specific H1 can interact with DNA not only in the major groove but also in the minor groove, and this induces changes in DNA structure. This DNA-protein interaction is specific for the sperm chromatin and may support the supercompact organization of the sperm chromatin.     

Altered histone-DNA interactions in rat liver chromatin containing 5-bromodeoxyuridine-substituted DNA

The extractability of the different histone types from rat liver chromatin was studied following the incorporation of bromodeoxyuridine (BrUdR) in liver DNA. This was accomplished by a continuous application of 20 mumol BrUdR/ml/h 17--41 after partial hepatectomy. As a result, thymidine (TdR) replacement by BrUdR of about 80% in the newly-synthesized DNA strand of approx. 30% of total liver DNA was obtained; this causes remarkable changes in the histone--DNA interactions as determined from the release of histones from liver nuclei by ammonium sulfate and ethidium bromide (EB), respectively. In particular, the relative amounts of the two slightly lysine-rich histones H2A and H2B remaining on the BrUdR chromatin proved to be about 3-fold higher than those remaining on the control chromatin of TdR-treated animals. Similarly, histones H1 and H3 tend to bind closer to BrUdR-containing DNA. These results may be of interst with regard to the well-known selective effects of BrUdR on differentiation processes.     

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.     

Interactions between arginine-rich histones and deoxyribonucleic acids. II. Circular dichroism

Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

Association products and conformations of salt-dissociated and acid-extracted histones. A two-phase procedure for isolating salt-dissociated histones

We present an extremely rapid and efficient method for the separation of salt-dissociated histones from DNA in which the macromolecular components of chicken erythrocyte chromatin are partitioned in a two-phase system of the water-soluble, nonionic polymers, poly(ethylene glycol) and dextran. We have compared the association products and conformations of salt-dissociated histones purified with the two-phase procedure and histones that had been extracted with 0.4 M H2SO4. In the gel chromatography system of D. R. vander Westhuyzen and C. von Holt (1971), FEBS Lett. 14, 333-337] the association products of salt-dissociated and acid-extracted histones are indistinguishable. Furthermore, the circular dichroism spectra of histones prepared with the two methods are identical within experimental error. These results indicate that histones extracted, with sulfuric acid can adopt conformations at least very similar to those of salt-dissociated preperties of total erythrocyte histones are the same in 2 M NaCl as those of these histones bound to DNA in chromatin in 1 mM Tris-Cl (pH 7.5). This result and the studies of Weintraub et al. [Weintraub, H., Palter, K., and Van Lente, F. (1975), Cell 6, 68-110] on the patterns of tryptic digest products of histones strongly suggest that in 2 M NaCl the histones exist in conformations very similar to their conformations when bound to DNA. The concept of native histone conformations is discussed in light of our results.     

Ethidium bromide as a probe of conformational heterogeneity of DNA in chromatin. The role of histone H1

The accessibility and the tertiary structure of the DNA inside chromatin were studied by using ethidium bromide (EB) as a fluorescent probe. The exclusion model of binding was refined by introductina a parameter alpha (0less than alpha less than 1) which measures the accessibility of the DNA and by taking into account when necessary the existence of two sets of binding sites. We were thus able to fit predicted and experimental isotherms and then to describe completely EB binding to native or partially histone depleted chromatin under various conditions. Itn native chromatin 95% of the DNA (alpha = 0.95) appears to be accessible to EB but two sets of sites are present. The first one corresponds to alpha = 0.13 and is characterized by an affinity constant which is higher by two orders of magnitude than that relative to pure DNA. The second set corresponds to alpha = 0.82 and the corresponding binding constant is only three or four times lower than that of pure DNA. The sites with high affinity are still present after treatment with formaldehyde but disappear after removal of histon H1. By comparison with chromatin treated with deoxycholate of with artifical complexes between H1 and DNA, high affinity sites were found only when all of the histons are bound to DNA. An alpha value around 0.8 is still obtained in 1 M NaC1 treated chromatin, pointing to the fact that histones H3 and H4 are preventing 20% of the DNA to intercalate EB.     

Assembly of DNA with histones and nonhistone chromosomal proteins in vitro

We have examined, by protein binding assays, thermal denaturation, and circular dichroism, the possible effects of histones on nonhistone chromosomal protein (NHCP) interactions with DNA. For these studies, we have fractionated mouse Krebs II chromosomal proteins into three discrete fractions: Mo, 5 M urea-soluble NHCP; M1, 5 M urea-1 M NaCl-soluble NHCP from 5 M urea-extracted chromatin; and M3, 5 M urea-3 M NaCl-soluble chromosomal proteins from 5 M urea-1 M NaCl-extracted chromatin. These fractions contain heterogeneous populations of NHCP, and were found to differentially affect histone binding to DNA by methods of reconstitution, or by direct binding of M0, M1, or M3 to urea-salt reconstituted DNA with histones. M0 was found to exert a unique effect on the thermal denaturation and circular dichroic spectra of DNA-histone complexes. M0 from Krebs II chromatin was also found to complete for DNA sites in the presence of M0 from mouse liver chromatin. In addition, in 5 M urea, pH 8.0, histone binding to DNA reached saturation at 1.85 mg/mg of DNA, higher than the in vivo ratio of 1.00 mg/mg of DNA. Saturation of histone binding to DNA occurred only in the presence of 5 M urea, resulting in a reduction of nonspecific histone-histone interactions on DNA.     

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.     

Studies on the role and mode of operation of the very-lysine-rich histones in eukaryote chromatin. Nuclear-magnetic-resonance studies on nucleoprotein and histone phi 1-DNA complexes from marine invertebrate sperm

Proton magnetic resonance and other measurements have been carried out in order to study the behaviour of the lysine-rich histones phi 1 in the sperm chromatin of certain marine invertebrates. Well defined particles (12 S) have been obtained from this chromatin by nuclease treatment. Chromatin solubility as a function of ionic strength shows a relaxation at salt concentrations higher than in the case of calf thymus nucleoprotein. Nuclear magnetic resonance (NMR) studies show that the release of histone from DNA occurs both in chromatin and in the reconstituted complexes at practically the same ionic strength as solubility relaxation. The higher the arginine content of a given phi 1, the higher the ionic strength at which both effects take place. The NMR results demonstrate that arginine residues are bound more strongly than lysine residues. The data overall show that phi 1 histones play a role in the contraction mechanism of sperm chromatin similar to that of H 1 histone in calf thymus chromatin. The highly contracted state of sperm chromatin is directly related to the increased arginine content of the phi 1 histone.     

Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.     

The in situ labeling of histone H3 in chromatin by a fluorescent probe

A sulfhydryl-specific fluorescent probe, N-3-pyrene maleimide, has been shown to label with high efficiency the sulfhydryl groups of histone H3 in nonsheared chromatin. The probe labels chromatin preparations obtained by mild homogenization or nuclease treatment of rat liver and mouse thymocyte, but not chick erythrocyte nuclei. Mononucleosomes from all nuclear preparations are labeled by the probe. The reaction is inhibited by prior reaction of the chromatin with N-ethyl maleimide. The reaction kinetics show fast and slow components representing reactions with cysteinyl sulfhydryl groups and lysyl epsilon-amino groups, respectively. Dissociation of the chromatin by urea (6 M) or sodium dodecyl sulfate (SDS) increases the fluorescence intensity (2-3 fold) and is maximal at approx. 0.01-0.02% (w/v) SDS. Histones extracted from the labelled chromatin show that approx. 80-90% of the label is associated with the histone fraction and column chromatography of this fraction shows that the label is primarily associated with histone H3. Labelling of the isolated histone fractions shows significant labelling only of histone H3. The intrinsic fluorescence of tryptophan is quenched by the labelled histone H3, but not by iodide, suggesting that non-histone (tryptophan-containing) proteins lie in close proximity to the labelled histone H3 but are not immediately accessible to external solvent. The labelled chromatin exhibits fluorescence anistropy, the anistropy parameter being 0.19 +/- 0.003 for chromatin, 0.05 +/- 0.01 for mononucleosomes and 0.0 for isolated histone H3. This demonstrates the restriction placed on the label's mobility by the chromatin fiber. The formation of a superhelix at 60-100 mM NaCl has been monitored with the probe. An increase in fluorescence intensity at 80 mM NaCl is observed with intact chromatin (but not H-1 depleted chromatin) followed by dissociation of the octamer in 1.50-2.0 M salt accompanied by a large increase in labelling.     

Mode of reconstitution of chicken erythrocyte and reticulocyte chromatin

The mode of reconstitution of chicken erythrocyte and reticulocyte chromatin has been investigated. Chromatin was dissociated in 2 M NaCl, 5 M urea, and 0.01 M potassium phosphate (pH 7.2) and was dialyzed against various NaCl concentrations in 5 M urea and 0.01 M potassium phosphate (pH 7.2). Histone reassociation to DNA occurs with the binding of histone H5 at 0.5 M NaCl in 5 M urea, followed by histone H1 at 0.4 M NaCl in 5 M urea. All the classes of histones are reassociated with DNA at 0.2 M NaCl in 5 M urea and binding of all classes of histones is complete in 0.1 M NaCl and 5 M urea. Nonhistone proteins reassociate with DNA before and at the same time that histones reassociate with DNA. Binding of nonhistone proteins to DNA appears to be complete in 5 M urea and 0.01 M potassium phosphate (pH 7.2). There is also found in both erythrocyte and reticulocyte chromatin a nonhistone protein present in relatively high concentrations, which remains associated with DNA in 2 M NaCl and 5 M urea. This tightly bound protein appears as one major band when chromatographed on sodium dodecyl sulfate-polyacrylamide gels, with a molecular weight of 95 000. This protein is soluble in phenol and sodium dodecyl sulfate but is insoluble in 5 M urea or 4 M guanidine hydrochloride. A fraction of reticulocyte nonhistone proteins was found to bind to DNA-cellulose in 5 M urea. The majority of these proteins elute at 0.15 M NaCl in 5 M urea but a significant fraction elutes at NaCl concentrations at which the bulk of the histones do not bind to DNA. The proteins that bind to free DNA have low molecular weights and do not show species speciificity. Approximatley 50% of the reticulocyte nonhistone protein does not bind to a DNA-cellulose column in 5 M urea and may require histones for complete reassociation.     

Histone neighbors in nuclei and extended chromatin

Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.