Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

Interaction of histone H1 molecules in a solution

Molecules of histones H1 isolated from the calf thymus, carp testicles and spermatozoa as well as trypsin-stable fragments of these proteins have been studied from the standpoint of their structure and interaction using methods of differential spectrophotometry, gel filtration and turbidimetry. The globular structure of histone H1 of the calf thymus is formed with an increase in the ionic strength of the medium and it is eluted as dimer with gel chromatography. With a considerable local increase of ionic strength (by addition of NaCl crystals) molecules of histones H1 form high-molecular aggregates from all the studied tissues. This aggregation is a result of interaction of globular trypsin-stable sites. Molecules of histone H1 from carp testicles and spermatozoa as well as their trypsin-stable fragments revealed no differences in the ability to form dimers and aggregates.     

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.     

Interaction of histone H1 from sea urchin sperm with superhelical and relaxed DNA

Complexes of histone H1 from sea urchin sperm (H1S) and calf thymus (H1T) with superhelical DNA I and relaxed circular DNA II have been analyzed by analytical sedimentation. Similar to H1T, the highly basic and relatively arginine-rich histone H1S preferentially interacts with DNA I compared to DNA II under competition conditions. However, H1S induces a stronger aggregation of bothforms of DNA than H1T. Below 0.05 M NaCl, the soluble complexes formed by both histones have similar properties, but aggregation proceeds in a different manner: H1S induces a stronger aggregation of DNA II as compared to DNA I, whereas H1T fails to aggregate DNA I.The results are explained on the basis of differences in amino acid sequence and structure of the two histones and related to the special chromatin condensing ability of histone H1S.     

Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 mm-Tris · HCl, 1 mm-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 mm-Tris · HCl, 1 mm-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Spatial organization of the (H3-H4-H2A-H2B)2 histone octamer

Structure of the (H2A-H2B-H3-H4)2 histone octamer isolated from calf thymus chromatin at ionic strength 0.1 to 4.0 M NaCl, pH 7.6, was studied spectrofluorometrically. Sensitivity of lambda max tyrosine fluorescence position to structural changes of histone oligomers and to the processes of their association was shown. It were detect two ranges of cooperative changes in histone optical parameters at 0.6-1.4 M NaCl (transition I) and at 2.4-3.4 M NaCl (transition II): Transition I corresponds to the formation of equilibrium system (hexamer) + (dimer) in equilibrium octamer. Transition II corresponds to the structural changes of the histone octamer. Thus, fluorescence anisotropy increases, lambda max for fluorescence spectrum is shifted to the longer wavelengths, contributions of two components to fluorescence decay change, a fraction of fluorescence accessible to the quenching by I- decreases. Histone octamer formation is characterized by making specific contacts between the (H2A-H2B) dimer and (H3-H4)2 tetramer. These contacts are realized at gradual changing of ionic strengths (by dialysis). In the case of abrupt local changes of the environment the process is irreversibly shifted to formation of unspecific high molecular aggregates. The important function role for energetically degenerated states of histone oligomers, energy barriers between which can be overcome by changing total conditions of histone microenvironment in chromatin is discussed.     

Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B

Experiments with antibodies induced by separated fragments 1-58 and 63- 125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

The absence of histone H1 from the chromatin fraction obtained by sonication of calf thymus nuclei under "quasiphysiological" ionic conditions.

The minor chromatin fraction was isolated from the sonicated calf thymus nuclei on the basis of its differential solubility in the "quasiphysiological" salt medium (0.1 M KCl-0.05 M NaCl-l mM MgCl2-1 mM CaCl2). Histone Hl is almost completely absent from this fraction. DNA isolated from this fraction occurs in three discrete low mol. wt. fragments. The fraction of chromatin which lacks histone Hl can also be obtained by two other methods. On of them consists in salt precipitation of the chromatin gel and its subsequent sonication. The second method includes precipitation of the sonicated chromatin gel by salts. In the first case the properties of the chromatin fraction which remains in the supernatant after centrifugation closely resemble those of the original salt-soluble nuclear fraction. The second method yields supernatant fraction also lacking histone Hl but containing heterogeneous DNA. Comparisons were also made of the sonically-solubilized nuclear fractions obtained in the complete salt medium and its mono and divalent cationic constituents.     

Isolation and physico-chemical properties of native histone complexes: dimer (H2-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2

The paper is concerned with the isolation of the native histone complexes: dimer (H2A-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2 from the calf thymus chromatin under soft conditions (hydroxyl apatite) fractionation with the subsequent gel filtration). Parameters of hydroxyl apatite saturation with chromatin are determined. The complexes obtained are free of DNA and nonhistone proteins. Absorption spectra parameters, quantum efficiencies and fluorescence spectra typical of the corresponding histone oligomers are established. Comparison of free tyrosine fluorescence spectra with histone tyrosyl ones revealed a long-wave shift in the latter.     

Laser Raman spectra of calf thymus chromatin and its constituents.

Extensive Raman measurements have been made on calf thymus chromatin, core chromatin, the (H3,H4)/DNA complex, and isolated DNA. The results indicate that the alpha-helical content of the nucleosomal histones gradually increases as they form the heterocomplexes that lead to the formation of the octameric nucleosome core. The secondary structure of the latter is not modified as it binds to DNA. The spectra indicate that the DNA essentially retains its B conformation in nucleosomes, although slight changes probably occur in the ribose-phosphate backbone. No specific interactions between the nucleosomal histones and DNA can be established from the spectra, but histone H1 possibly interacts selectively with the thymine bases.     

Nucleosomes are assembled into discrete size structures by histone H1 in vitro

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller supernucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supernucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.     

Non-histone chromosomal proteins easily extractible from chick erythrocytes.

Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryote chromatin. The properties of the N-terminal and C-terminal halves of histone H1.

Restricted chymotrypsin digestion of calf thymus H1 histone gives two fragments, residues 1--106 and 107--C-terminal. These were studied by proton magnetic resonance and circular dichroism. The N-terminal fragment exhibited some salt-induced structure in aqueous solution, but this did not parallel the globular structure of the intact H1 molecule. Comparison of circular dichroism results with helix predictions for this portion of the molecule suggests that the secondary structure may be the same in this fragment as it is in the corresponding region of the whole molecule. The C-terminal fragments show very little salt-induced structure. The N-terminal fragments binds to DNA very weakly, but the C-terminal fragment binds as strongly as the whole molecule. In the C-terminal fragment, about one quarter of the lysine residues are not bound to the DNA in water, but initial increase of salt concentration causes them to become bound. This increasing binding occurs under the same ionic conditions that cause chromatin condensation and condensation of H1 - DNA complexes, and it is suggested that there may be a connection between these phenomena.     

The effects of daunomycin antibiotic on histone H(1): thermal denaturation and fluorescence spectroscopy studies

Using thermal denaturation and fluorescence spectroscopy, we have investigated the interaction of antitumor antibiotic, daunomycin, with calf thymus histone H(1) under several ionic strengths. The results show that daunomycin binds to histone H(1) and increases its melting temperature. Increasing ionic strength elevates this effect. Fluorescence emission data show that the interaction of daunomycin with histone H(1) decreases the emission intensity at 325 nm and induces hyperchromicity in the emission spectrum of the drug. The results suggest that histone H(1) can be considered as a new target for drug action at the chromatin level.     

On the interactions of histone H4 and H4 peptides with DNA. Electrooptical, hydrodynamic and electron microscopy studies

The interactions of DNA with histone H4 and with its fragments N-H4 (1-84) and C-H4 (85-102) have been studied by using electrooptical techniques, viscosity and electron microscopy. Electron microscopy reveals that histone H4 induces a large folding of DNA molecules : this is in agreement with electrooptical measurements which indicate that, with the increase of their ratio, H4/DNA complexes undergo a gradual process of condensation. Viscosity measurements show that complexes at ratios up to 0.20-0.25 become more rigid as compared to DNA. It appears that C-H4, and not the N-H4 fragment, causes a great distorsion to the structure of DNA, accompanied by an increase of rigidity at ratios up to 0.20-0.25, as occurs for H4/DNA complexes. Electrooptical studies of C-H4/DNA complexes show, along a range of histone/DNA ratios, an important permanent dipole component. These effects reveal a particular mode of interaction of C-H4 with DNA, indicating that some charged residues of the peptide are kept distant enough from the DNA backbone. As no dipole character, in addition to that shown for DNA, has been detected for H4/DNA complexes, it is concluded that the conformation of the H4 molecule modifies to some extent the interaction of the C-terminal region. Our results show that this histone, and particularly its C-terminal region, is important as a determinant factor in the folding of DNA within artificial complexes.