Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA.

Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA.     

The structure of chromatin from the pigeon brain. II. Sedimentation analysis of oligonucleosome density

Compaction of pigeon brain and rat thymus chromatin differing in the length of the linker DNA has been studied by the method of velocity sedimentation. The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain in solution of different ionic strength (0.005-0.085) has been analyzed. The analyses of these dependences showed that the structure of oligonucleosomes of both cell types at low ionic conditions may be described by the model of a zig-zag-shaped nucleosomal chain. The process of compaction of the oligonucleosomes at higher ionic strength (0.045-0.085) proceeds similarly for brain and thymus chromatin. The formation of a superhelical structure is determined by the interaction of no less than 6 nucleosomes; the compactness of the structure is significantly increased when the number of nucleosomes in the chain exceeds 10. The ability of the brain oligonucleosomes to form a compact structure despite the short linker allow the suggestion that in brain short chromatin the DNA chain does not form two complete turns in the nucleosome. This provides necessary flexibility of brain chromatin.     

Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.     

Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.     

A stable alpha-helical element in the carboxy-terminal domain of free and chromatin-bound histone H1 from sea urchin sperm.

The carboxy-terminal domain (residues 121-248) of sea urchin sperm-specific H1 is not random coil but partly alpha-helical, even in 1 mM sodium phosphate, pH 7. The helix resides in a 57 residue proline-free segment which, in the intact histone, immediately abuts the central globular domain. The proline-free region, which is rich in lysine and alanine, is relatively resistant to tryptic digestion when the carboxy-terminal domain is bound to DNA. Two (overlapping) resistant peptides are shown by circular dichroism measurements to be substantially alpha-helical in 1 mM sodium phosphate and to increase in helix content to approximately 70% in 1 M NaCLO4. Tryptic digestion of chromatin gives resistant fragments containing both the globular domain and the contiguous proline-free segment, strongly suggesting that the alpha-helical segment also exists in chromatin, where it would be ideally placed to direct the path of the linker DNA entering or leaving the nucleosome. The linker in sea urchin sperm chromatin is long (approximately 74 bp), and the unusually long alpha-helical segment in the carboxy-terminal tail of sperm H1 which has amphipathic character due to the alanine distribution, and is likely to be curved, may be a special feature tailored to organize it.     

Structural transformations of oligonucleosomes from pigeon erythrocyte chromatin

The methods of velocity sedimentation and circular dichroism have been used to investigate structural rearrangements of pigeon erythrocyte oligonucleosomes isolated after digestion with micrococcal nuclease (oligonucleosomes-M) or pancreatic DNase I (oligonucleosomes-D), in the wide range of ionic strength (mu from 0.005 to 0.5). The electrophoretic analysis of DNA isolated from the oligonucleosomes has revealed internal cuts in the DNA chain of oligonucleosomes-D. In spite of this fact the conformational parameters of DNA in both types of oligonucleosomes are practically indistinguishable, and their optical and hydrodynamic properties vary in a similar way with increasing ionic strength of the solution. The specificity of DNase I action results in the ability of oligonucleosomes-D to form homogeneous associates at mu = 0.065, which seems to be due to the existence of elongated intact ends of linker DNA in oligonucleosomes-D. It has been shown that the integrity of oligonucleosomes-D in a wide range of ionic strength is maintained by histones H1 and H5, because after their dissociation the sedimentation coefficient sharply decreases. The results obtained reveal the multifunctional role of lysine-rich histones and intact linker in the processes of compaction and association of oligonucleosomes.     

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.     

Role of the histone "tails" in the folding of oligonucleosomes depleted of histone H1.

An oligonucleosome 12-mer was reconstituted in the absence of linker histones, onto a DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Ly-techinus variegatus (Simpson, R. T., Thoma, F. S., and Burbaker, J. M. (1985) Cell 42, 799-808). The ionic strength-dependent folding of this nucleohistone complex was compared with that of a native oligonucleosome fraction obtained from chicken erythrocyte chromatin, which had been carefully stripped of linker histones and fractionated in sucrose gradients. The DNA of this native fraction exhibited a narrow size distribution centered around the length of the 208-12 DNA template used in the reconstituted complex. These two complexes displayed very similar hydrodynamic behavior as judged by sedimentation velocity analysis. By combining these data with electron microscopy analysis, it was shown that the salt-dependent folding of oligonucleosomes in the absence of linker histones involves the bending of the linker DNA region connecting adjacent nucleosomes. It was also found that selective removal by trypsin of the N-terminal regions ("tails") of the core histones prevents the oligonucleosome chains from folding. Thus, in the absence of these histone domains, the bending of the linker DNA necessary to bring the nucleosomes in contact is completely abolished. In addition to the complete lack of folding, removal of the histone tails results in an unwinding at low salt of a 20-base pair region at each flanking side of the nucleosome core particle. The possible functional relevance of these results is discussed.     

Linear dichroism of chromatin fibers

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Friend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations by the use of flow linear dichroism and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA--2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA--for chromatins with linker DNA 10-30 b.p. it is partially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric fields do not have an effect on chromatin structure, while higher electric fields (more than 7 kV/cm) distort the structure of chromatin.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

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.     

Structural properties of barley nucleosomes

The structural properties of barley oligonucleosomes are investigated and compared to those of rat liver oligomers. Extraction of barley chromatin was performed using mild nuclease digestion of isolated nuclei leading to a low ionic strength soluble fraction. Oligonucleosomes were fractionated on sucrose gradients and characterized for DNA and histone content. Physico-chemical studies (sedimentation, circular dichroism and electric birefringence) showed that barley oligonucleosomes exhibit properties very close to those of the H1-depleted rat liver counterparts. Moreover, in situ, barley linker DNA was more sensitive to micrococcal nuclease digestion than that of rat liver. These results suggest that barley oligonucleosomes show a less compact structure than their rat liver counterparts and appear to be in contradiction with the very condensed organization of barley chromatin previously suggested.     

Optical Anisotropy of Chromatin. Flow Linear Dichroism and Electric Dichroism Studies

Abstract

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentraitons by the use of flow linear dichroism (LD) and electric dichroism.

We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA—2 mM NaCl and close positive values in the range of 2–100 mM NaCl. Mg²⁺ cations, in contrast to Na⁺ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5–10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers.

The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA—for chromatins with the linker DNA of 10–30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible.

Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin.

Presented resutls explain the contradictory data obtained by electrooptical and hydrooptical methods.     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

Characteristics of the structural state of DNA in chromatin with short linker

Various fragments of pigeon brain neuron chromatin with very short linker DNA have been studied by circular dichroism (CD). The DNA structure in core particles of the brain and thymus chromatins is very similar. Linker DNA and a part of core DNA in the mononucleosomes of brain chromatin is extended. This conclusion follows from increasing CD amplitude of the brain mononucleosomes as compared with the corresponding value for thymus mononucleosomes. The internucleosome interactions stabilized the compactness of core DNA in the brain oligonucleosomes. The whole linker DNA of the brain chromatin unlike thymus chromatin is extended at low ionic strength. This fact can explain the brain chromatin ability to form a compact structure with increasing ionic strength.     

Phosphorylation of histone variant regions in chromatin: unlocking the linker?

Histone variants illuminate the behavior of chromatin through their unique structures and patterns of postsynthetic modification. This review examines the literature on heteromorphous histone structures in chromatin, structures that are primary targets for histone kinases and phosphatases in vivo. Special attention is paid to certain well-studied experimental systems: mammalian culture cells, chicken erythrocytes, sea urchin sperm, wheat sprouts, Tetrahymena, and budding yeast. A common theme emerges from these studies. Specialized, highly basic structures in histone variants promote chromatin condensation in a variety of developmental situations. Before, and sometimes after condensed chromatin is formed, the chromatin is rendered soluble by phosphorylation of the heteromorphous regions, preventing their interaction with linker DNA. A simple structural model accounting for histone variation and phosphorylation is presented.