Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Interaction of dipalmitoyl-phosphatidylcholine with calf thymus histone H1

The interaction between dipalmitoyl-phosphatidylcholine and calf thymus histone H1 has been studied. A protein-phospholipid complex, resulting from this interaction, has been isolated by centrifugation in a sucrose gradient. The phospholipid-histone interaction causes an increase in the alpha-helix content of the protein; the corresponding conformational transition is observed by CD studies in the far-u.v. region. The only tyrosine residue of the protein can be advantageously used as an intrinsic fluorescent probe; thus, fluorescence spectra indicate that protein folding induced by phospholipids is concomitant with the tyrosine transfer into a more hydrophobic environment. The trypsin-resistant core of the histone is also folded in the presence of the phospholipid but the conformational transition occurs at lower lipid concentration than for the intact protein. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicates that the protein shifts the transition temperature of the phospholipid from 41.5 to 44.0 degrees. Secondary structure prediction of the trypsin-resistant core of the histone indicates the existence of an amphipathic helix that could be responsible for the lipid-protein interaction.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

Characteristics of the tertiary structure of histone H1 from the calf thymus

By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4.     

Effect of ionic strength of the medium on the structure and aggregation of histone H1 from the calf thymus

In the region of 50 mM NaCl histone HI has the structure with a great number of binding sites with fluorescent probe 1.8-ANS as compared to the structure formed in solution of 0.6-1.0 M NaCl. These sites, however, have a lower constant of binding with the probe and are characterized by a higher surrounding polarity. At local significant increase of the ionic strength histone HI molecules form stable oligomers having hydrophobic cavities. A conclusion is made about the importance of cationic envelope formed by N- and C-ends around the globular "head", for manifesting effective interactions between several molecules of histone HI.     

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.     

Effect of urea on proteolysis of histone H2A in calf thymus nuclei

The 1 hour incubation of calf thymus nuclei at 37 degrees C leads to a proteolysis of the histones H1, H3 and H2B. Urea does not influence the histone degradation while 1.5 and 2.0 M NaCl lead to the proteolysis of the H2A histone. On this background, 2 M urea restrains the degradation of the H2A histone. It is assumed that hydrogen bonds are very important for the activity of the proteinases and its interaction with the H2A histone.     

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.     

A quantitative analysis of histone H1 in rabbit thymus nuclei.

The relative quantity of histone H1 in rabbit thymus whole histone was determined to be 17.2% (w/w). This implies that there is, on average, one histone H1 molecule per nucleosome.     

Phosphorylation of calf thymus H1 histone by calcium-activated,phospholipid-dependent protein kinase

Ca²⁺-activated, phospholipid-dependent protein kinase recently found in mammalian tissues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) $\text{J.}$$\text{Biol.}$$\text{Chem.}$$\text{254}$, 3692–3695) is able to phosphorylate five fractions of calf thymus histone. H1 histone serves as a preferential substrate, and approximately two moles of phosphate are incorporated into every mole of this histone. Analysis on the N-bromosuccinimide-bisected fragments of this radioactive histone has revealed that the enzyme phosphorylates preferentially seryl and threonyl residues located in the carboxyl-terminal half of this histone molecule.     

Modification of histone binding in calf thymus chromatin and in the chromatin-protamine complex by acetic anhydride.

A relationship between side-chain modification of histones and their displaceability from DNA has been investigated using calf thymus chromatin which was chemically acetylated with acetic anhydride. When the chromatin is treated with increasingly higher concentrations of the reagent, histones become acetylated to an increasingly greater extent, attaining the modification at 23-24 sites for histone I, 5-6 for IIb1, 9-10 for IIb2, 5-6 for III and 3-4 for IV. As the chromatin becomes more acetylated, NaCl concentrations required for histone removal are lowered. Saturation binding of protamine does not bring about either an increase in the number of acetylation sites of histones in chromatin or a decrease of the NaCl requirement for dissociation of the acetylated chromatins. A comparison of the present results with the extents of histone acetylation known to occur enzymatically in vivo indicates that the complete removal of somatic histones during transformation of chromatin in spermiogenesis cannot be explained on the basis of decreased binding of the histone to DNA by acetylation or by a combination of acetylation and protamine binding, suggesting that the displacement process may require some additional processes.     

The quantitative protein composition of calf thymus chromatin.

The proteins of calf thymus chromatin were analysed quantitatively using a combination of polyacrylamide and cellogel electrophoreses. Quantification was achieved by determining the staining values of each purified histone with amido-black. The results indicate that, on average, 700 molecules histone F1, 850 of F2al, 1440 of F2a2, 1 890 of F2b, 2380 of F3 and 500-1000 non-histone molecules are bound per 10(5) base pairs of DNA. This suggests a moderately dense protein covering of the DNA.     

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.     

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.