Erythroid-specific gene chromatin has an altered association with linker histones.

The chromatin of several genes was assayed for sensitivity to DNAase I and for solubility as polynucleosomes in 0.15 M NaCl. The degree of solubility of chromatin fragments as polynucleosomes in 0.15 M NaCl correlates well with the sensitivity to DNAase I for several genes. Chromatin of repressed, housekeeping and erythroid-specific genes can be distinguished as distinct groups by the degree to which they display these properties. NaCl precipitation of chromatin fragments stripped and then reconstituted with varying quantities of H1 and H5 (linker) histones indicate that the polynucleosomes of erythroid-specific genes have altered interaction with these histones. Linker histones interacted with bulk chromatin and in the chromatin of the repressed ovalbumin and vitellogenin genes to form salt precipitable structures. Chromatin of erythroid-specific genes (histone H5 and beta-globin) as well as that of the histone H2A.F gene was resistant to linker histone induced precipitation.     

Reconstitution of compact polynucleosomes and comparison of the functions of histones H1 and H5

Polynucleosomes with a definite length (about 4,500 base pairs) were prepared from chicken erythrocyte nuclei without depleting magnesium ions from the medium. The polynucleosomes in the presence of Mg2+ ions as well as monovalent salts were more compact than those with monovalent salts alone. We minimized the occurrence of nicks in the DNA of nucleosome fiber during the preparation. When histones H1 and H5 were completely removed from polynucleosomes, linker histone-depleted polynucleosomes sedimented slower than the original ones. When isolated histone H1 or H5 was reassembled with linker histone-depleted polynucleosomes, no significant difference was observed among the reconstituted polynucleosomes with histone H1, the reconstituted polynucleosomes with histone H5, and the original polynucleosomes. We concluded that histones H1 and H5 are similar in their effects on higher order structure of polynucleosomes, as far as can be judged from such characteristics as sedimentation velocity, linker histone content, and the patterns of nuclease digestion.     

Quantitative determination of linking number differences between circular polynucleosomes and histone H1-bound circular polynucleosomes

With the aim of discovering contribution of histone H1 to linking number changes of DNA, determination of linking number differences between histone H1-free circular polynucleosomes and histone H1-bound circular polynucleosomes was carried out during our investigations. Our results showed that on average, binding of ～11.5 histone H1 molecules causes one linking number change in circular polynucleosomes in the presence of 1.5?mM spermidine. When concentrations of spermidine decreases or increases, these linking number differences decrease significantly. It is therefore evident that linking number changes caused by histone H1 are spermidine concentration-dependent.     

The specificity of human autoantibodies that react with both cell nuclei and plasma membranes: the nuclear antigen is present on core mononucleosomes.

We have examined the nature of the nuclear antigen recognized by certain natural human antibodies that react specifically with both cell nuclei and plasma membranes from many species. Partial purification of these antibodies, called X-ANA, is achieved by binding to and rapid elution from the surface of viable human leukocytes. Chicken erythrocyte chromatin was solubilized by digestion with staphylococcal nuclease and fractionated into a 0.15 M NaCl soluble fraction that consisted of core mononucleosomes lacking H1/H5, and a 0.15 M NaCl insoluble fraction composed of polynucleosomes with H1/H5 present. No proteins other than histones were detected. Native and reconstituted mononucleosomes displaced IgG of the leukocyte eluates from nuclei of frozen mouse kidney sections and from the walls of plastic tubes coated with polynucleosomes. The reconstituted core mononucleosomes were 4- 10-fold less efficient inhibitors than native mononucleosomes. Trypsin digested mononucleosomes, free high m.w. DNA, and free histones displayed no or very weak inhibitory activity. The data indicate that X-ANA recognize a complex consisting of the core histones H2A, H2B, H3, H4, and DNA of 140 to 200 base pairs in length.     

Exchange of H1 histone depends on aggregation of chromatin, not simply on ionic strength

Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.     

Differences among chicken erythrocyte histones H1 and H5 in associating with H1-depleted polynucleosomes

1. Chicken erythrocyte histones H1a, H1b and H5 were associated to H1-depleted polynucleosomes from rat liver and the products were probed by digestion with micrococcal nuclease. 2. The digestion response of complexes with purified H1a or H1b resembled that of native polynucleosomes, while the digestion response of complexes with purified H5 exhibited specific different features--but none of these complexes resembled those with unfractionated histones H1a, H1b and H5. 3. However, after mixing purified histones H1a, H1b and H5 in the proportions (0.15:0.25:0.9) as these occur in erythrocyte nuclei and associating this mixture, the digestion response of the complexes was similar to that of the complexes with unfractionated histones.     

Rearrangement of chromatin structure induced by increasing ionic strength and temperature.

Native rat liver chromatin fragments exposed to 600 mM NaCl at 37 degrees C for 45 min exhibit substantial modification of their original (approximately 200 base pairs) repeating subunit structure: a new repeat of 140 base pairs, superimposed on a high background, is observed after micrococcal nuclease digestion. The same material appears, in the electron microscope, as clusters of tightly packed beads connected by stretches of 'free' DNA. These modifications are not observed when the native chromatin is incubated at 37 degrees C at NaCl concentrations up to 400 mM. When native rat liver chromatin depleted of histone H1 by tRNA extraction is exposed to ionic strengths up to 600 mM NaCl at 4 degrees C, almost no modifications of the original native repeating structure are observed. However, when the incubation is carried out at 37 degrees C in 150, 300 or 400 mM NaCl, rearrangements of the native structure occur as indicated by micrococcal nuclease digestion and electron microscopic studies. Incubation of H1-depleted chromatin at 600 mM NaCl for 45 min at 37 degrees C induces, as for the native chromatin, a complete rearrangement characterized by the appearance of a 140-base-pair repeat superimposed on a high background upon digestion by micrococcal nuclease. It is suggested that these rearrangements are mediated by hydrophobic interactions between the histone cores and are prevented at ionic strengths lower than 500 mM by the presence of histone H1.     

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.     

Exchange of proteins during immunofractionation of chromatin

The migration and rearrangement of chromosomal proteins during immunofractionation of chromatin has been investigated. Oligonucleosomes from two different chromatins, chicken erythrocyte or rat liver, were mixed with oligonucleosomes from the other species which had been depleted of histones H1/H5 and high mobility group proteins (HMGs). The mixture was treated with buffers of various ionic strengths and immunofractionated on an anti-H1 degrees/H5 or anti-HMG-17 IgG-Sepharose column. The type of DNA, which was retained as the bound fraction on the column, was determined by slot blot analysis using nick-translated repetitive DNA probes from either chicken or rat. The results indicate that in low ionic strength buffers (i.e., below 40 mM NaCl), there is very little exchange of either histone H5 or HMG-17 among nucleosomes and therefore we suggest that it is possible to fractionate nucleosomes according to their antigenic content.     

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.     

The distribution of H1 histone is nonuniform in chromatin and correlates with different degrees of condensation

Salt induces aggregation of large chromatin fragments maximally at 150-200 mM NaCl. The soluble fragments are depleted of H1 histones while the aggregated fragments are enriched. H1 histones did not equilibrate between the soluble and insoluble chromatin fractions when they were recycled through the process of salt-induced aggregation. The chromatin fragments that resisted aggregation retained more H1c subtype than they did H1 ab, correlating with previous results which showed complexes of H1c with DNA resisted salt-induced aggregation much more than complexes of DNA with other subtypes. The chromatin that was soluble at physiological concentrations of NaCl was DNase I sensitive and enriched in acetylated core histones. We conclude that H1 histone is nonuniformly distributed in chromatin in a stable pattern that probably correlates with the different degrees of condensation known to exist in vivo.     

Differences in the binding of H1 variants to DNA. Cooperativity and linker-length related distribution

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding. 1. All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod-like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1. 2. The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well-defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM. 3. The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.     

Porcine follitropin. The amino-acid sequence of the beta subunit

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.     

Anomalous nuclease digestion of Holothuria sperm chromatin containing histone H1 variants

We have investigated the micrococcal nuclease digestion of chromatin from the spermatozoa of the sea cucumber Holothuria tubulosa. This chromatin contains minor protein variants related to histone H1 with a high proportion of basic amino acids. One of these variants, protein phi 0, represents about 4% of the total histones. It is 78 amino acids long and its amino acid composition and sequence are related to the very basic C-terminal region of histone H1. The presence of these proteins induces an unusual digestion pattern. Oligonucleosomal particles which are soluble at 150 mM NaCl are depleted of protein phi 0 and they are also defective in histone H1. A low percentage of the insoluble material can be solubilized at lower NaCl concentrations (50 mM). These oligonucleosomal particles show a very peculiar protein content, since at early digestion times, they contain histone H1 and protein phi 0 exclusively. We conclude that these particles arise from a cooperative displacement of core histones by protein phi 0 and histone H1. These results show that minor changes in histone H1 complement can result in the formation of artifactual particles upon microccocal nuclease digestion. These observations may be of interest in other systems which contain H1 variants.     

The higher order repeat structure of chromatin is built up of globular particles containing eight nucleosomes

Mild nuclease digestion of rat liver chromatin generates particles with sedimentation coefficients of about 33S, 60S, and 90S (in 50 mM NaCl). The kinetics of appearance and disappearance of these particles with progressive digestion suggest that they are produced by cleavage from a higher order repeat structure, the 33S particle representing the monomer. At an intermediate stage of digestion, about 75 % of the nuclear chromatin can be recovered as monomers to trimers of this higher order structure. Sedimentation profiles indicate that monomer particles containing 7–8 nucleosomes occur at the highest frequency. The DNA fragments in monomers have a size corresponding to hepta- and octanucleosomes, and those in dimers have a size corresponding to chains of sixteen nucleosomes. The higher order repeat structure is only stable between 30 and 200 mM NaCl; the particles unfold below 30 and above 200 mM NaCl. When examined by electron microscopy, monomers and dimers appear as compact globular structures. Relaxation by lowering the salt concentration results in the appearance of polynucleosomes with a chain length of eight beads in the monomer and sixteen in the dimer particle. These results indicate that the unit particle of the higher order repeat structure of rat liver chromatin contains eight nucleosomes.     

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.     

Synchrotron X-ray scattering study of chromatin condensation induced by monovalent salt: analysis of the small-angle scattering data

Small-angle X-ray scattering experiments were carried out on rat thymus chromatin in "native" and "H1-depleted" states at various NaCl concentrations using synchrotron radiation. From the analysis of cross-sectional Guinier plots, the radius of gyration of the cross section (Rc) and the mass per unit length (Mc) of native chromatin were evaluated. In the absence of NaCl, the cross section of chromatin filament has a radius of gyration of 3.44 nm, suggesting the structure corresponding to the "10 nm" filament. With increasing NaCl concentration, the Rc value increases steeply to 6.74 nm at 5 mM NaCl and then gradually to 8.82 nm at 50 mM NaCl, whereas the Mc value, which is determined relative to that of tobacco mosaic virus (TMV), increases steadily from 1.58 nucleosomes per 10 nm in the absence of NaCl to 7.66 nucleosomes per 10 nm at 50 mM NaCl. However, since calibration with TMV tends to overestimate the Mc value, the actual Mc values may be less than those values. Above about 40 mM NaCl, aggregation of chromatin is suggested. Similar analysis of H1-depleted chromatin confirmed that H1-depleted chromatin takes a more disordered structure than native chromatin at low ionic strength and does not undergo a definite structure change upon further addition of NaCl.