Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.     

Generation of a third-order folded structure for chromatin

A model for the initiation of the diffuse-condensed transition of chromatin induced by a change in the conformation of lysine-rich histones is proposed. Three levels of folded structures are discussed. The first-order folded structure refers to the structure of the repeat unit of chromatin, which is called the nucleosome. The nucleosome contains a nuclease resistant region in which 140 base pairs of DNA are wrapped around the surface of a histone aggregated of two copies each of the histones H2A, H2B, H3 and H4. This DNA-histone aggregate is called a core particle. The nuclease accessible region of the nucleosome is approximately 60 base pairs of DNA which link the core particle, hence the terminology “linker DNA.” The lysine-rich histones, (Hl, H5), which are more loosely bound than the core histones, are associated with the linker DNA. The second-order folded structure refers to the conformation of a polynucleosome. Based on neutron scattering and quasielastic light scattering studies the second-order folded structure is assumed to be an extended helix in solution with 5–7 nucleosome units per turn. The third-order folded structure is defined as that structure resulting from the first stage in the condensation process induced by a conformational change in the lysine-rich histones. Generation of the third-order folded structure in the proposed model is effected by an increased affinity of the lysine-rich histones for super-helical DNA in the core particles in adjacent turns of the second-order folded structure. Since the lysine-rich histones preferentially bind to A-T rich regions in DNA, the distribution of these regions would determine the third-order folded structure. The net effect of a non-random distribution of A-T rich regions as in the proposed model is the generation of a helix for the third-order folded structure. The assumption of a non-random distribution of A-T rich regions is indirectly supported by proflavine binding studies reported herein and by the existence of repetitive and non-repetitive DNA regions inferred from renaturation studies. One consequence of the proposed mechanism is that the majority of the A-T rich regions are in the interior of the third-order folded structure. Promoter sites of high A-T content would then be inaccessible to polymerases. The proposed model also suggests a role for spacer DNA in the genome. Higher order folded structures must also be present in the final state of condensed chromatin since the three orders of folded structures considered in this communication accounts for only 2% of that required in the diffuse-condensed transition.     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

Histones from nucleated erythrocytes of the marine invertebrate Sipunculus nudus

Total and lysine-rich histones were extracted from purified sipunculid erythrocyte nuclei with 0.25 N HCl and 0.74 N perchloric acid, respectively. The histones were fractionated and purified by gel filtration and ion exchange chromatography. Sipunculid PCA extract shows three lysine-rich histones, one of which may be a fraction specific to erythrocytes. The histones H₃ and H₄ of this marine invertebrate are very similar to their vertebrate homologues. Whereas no fraction H_2B has been detected in our investigation, the fraction H_2A has an unusual chromatographic behaviour if compared to its vertebrate homologue. These features may be the reflection of a peculiar distribution of histones in the chromatin of sipunculid erythrocytes.     

The same amount of DNA is organized in in vitro-assembled nucleosomes irrespective of the origin of the histones.

The four histones H2A, H2B, H3 and H4 from calf thymus, CHO and sea urchin gastrula cells were associated by stepwise dialysis from 2 M NaCl with SV40 DNA Form I. The in vitro-assembled chromatins were visualized by electron microscopy and the size of the DNA fragments generated by digestion with DNase II was determined. Irrespective of the origin of the histones, the size of the smallest DNA band generated at early times of digestion was about 190 base pairs, whereas oligomeric DNA bands were multiples of 140 bp. These results support our previous proposal that the four histones H2A, H2B, H3 and H4 are able to organize more than 140 bp of DNA, but do not provide any evidence that the variability of histones H2A and H2B plays a role in the variability of the DNA repeat length of native chromatins.     

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

Studies on DNA bound to histones in chromatin]

Regions of DNA protected by histones against the action of DNAse 1 in the chromatin were isolated. Such DNA fragments ("subhistones" DNA) have 80% double helix structure, their nucleotide composition is close to that of total DNA, and their sedimentation constant is within the range of 2-2.7S for completely denatured molecules. Kinetics of renaturation of "subhistone" DNA was studied: within a wide range of Cot values, renaturation curves of total and "subhistone" DNA are almost identical. According to the data on hybridization with nuclear d-RNA, "subhistone" DNA is transcribed in the cell. The data obtained witness for uniform character of distribution of histones along the DNA chain in the chromatin. DNA sites which are active in RNA synthesis seem to be bound to histones as well as the non-active ones. No significant difference was found in the hybridization of "subhistone" DNA from rat liver and thymus with ibver nuclear RNA.     

Predictability of sequence homologies among lysine-rich histones by immunological distance

The relationship between immunological distance (I.D.) measured by microcomplement fixation and amino acid sequence difference for lysine-rich histones was tested using antisera to lysine-rich histones of known sequence, chicken H1 and H5, goose H5, and trout H1 as well as to trout H5. The best relationship between I.D. (y) and percent sequence difference (x) for lysine-rich histones, y = 2x, applies as well to other histones of known sequence but it differs from y = 5x, reported for other proteins and often used for histones. Although deviations indicate that I.D. is a poor predictor of primary sequence differences among histones, it suggests that trout H5 is more closely related to H1 than to chicken H5.     

Inhibition of mammary gland cyclic AMP-dependent protein kinase by arginine-rich histones.

The cyclic AMP-dependent protein kinase activity from lactating bovine mammary gland efficiently phosphorylates lysine-rich histones but not arginine-rich histones. It is shown that arginine-rich histones in fact inhibit phosphorylation of lysine-rich histones. Polyarginine and a range of low molecular weight cationic molecules are also inhibitors. Inhibition of histone H2b phosphorylation by histones H4 and H3 is competitive with respect to H2b. This inhibition behaviour may be tissue-specific since the protein kinase activity in crude extracts from lactating bovine mammary gland, although heterogeneous, may be completely inhibited (>95%) by arginine-rich histones and polyarginine.     

H1 histone variants in Xenopus laevis

The H1 histones from erythrocytes, livers, intestines, testes, and embryos of Xenopus laevis have been examined electrophoretically. This species has been found to contain at least five electrophoretically resolvable lysine-rich histones in addition to the presumptive H5 histone of erythrocytes. Quantitative and qualitative distinctions between the H1 histones from each source were readily observed. Three H1 histones (H1A, H1B, and H1C) were found in both embryos and adult tissues, although in varying amounts. Two other H1 histones (H1D and H1E) were found only in adult tissues. Comparative SDS gel V8 protease cleavage maps of the lysine-rich histones from testes and erythrocytes have demonstrated that the “adult-specific” H1D and H1E are not artifacts of proteolysis and may be closely related to the presumptive H5 histone. Spermatogenic cells were found to be similar to embryonic cells in being deficient in H1D and H1E. These observations suggest that H1D and H1E are enriched in cell types with low rates of cell division similar to the mammalian H1° histone. The results presented here demonstrate a previously unrecognized degree of developmental and cell-specific variance in the H1 histones of Xenopus laevis.     

Reactivity of heterogeneous F1 histones with glutaraldehyde and formaldehyde

The rates of glutaraldehyde and formaldehyde fixation of F1 histones have been investigated using chromatin from rat pancreas, chicken erythrocyte, and human spleen. These chromatins differ in number, type and relative proportion of F1 species present. In all cases the rates of fixation by glutaraldehyde and formaldehyde of the F1 components is much faster than for the other histones. The rates of fixation of F1-type histones are similar in each case with the exception of one minor F1 histone from chicken which reacts slower than the rest of that F1 group. The results suggest that the interactions of all F1 type histones with DNA are similar.     

Molecular cloning of a pea H1 histone cDNA

A pea (Pisum sativum, var. Little Marvel) H1 histone cDNA has been isolated from a lambda gt11 expression vector library. This cDNA has been sequenced and shown to represent the entire protein-coding region of the mRNA. The deduced protein sequence is 265 amino acids long (28018 Da) and contains 70 lysines and 3 arginines. The structure of the encoded protein is comparable to animal lysine-rich histones. The central region, which has an amino acid composition similar to that found in the globular domains of animal lysine-rich histones, is flanked by an amino-terminal region rich in lysine, glutamic acid and proline and by a carboxyl-terminal region rich in lysine, alanine, valine and proline. Despite the structural similarities, the protein has little sequence homology with animal lysine-rich histones. This H1 protein is unusual because 12 of the first 40 amino acids are glutamic acid.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

Histones with high lysine content

1. The preparation and properties of lysine-rich histones, which differ in a number of respects from the classical arginine-rich histones, have been described. 2. Lysine-rich histones, like those previously known, are located in cell nuclei. 3. Lysine-rich histones dissociate more readily from combination with nucleic acid than do other histones.     

Chromatin-associated proteins of the developing sea urchin embryo. II. Acid-soluble proteins

Of the five well-characterized histories, only the slightly lysine-rich histories F2a and F2b are present in sea urchin embryos before the 16-cell stage. At the 16-cell stage, the arginine-rich (F3) and lysine-rich (Fla) histones appear and all the major histones are then present in the same relative proportions until the pluteus stage except for a second lysine-rich protein, Flb, which is first detected at 12 to 16 hours of development and increases to the pluteus stage. From 16 cells to pluteus at 70 hours, all the histones are labeled by a 30-minute incubation with radioactive lysine, with the exception of the lysine-rich histone Fla which does not incorporate label after 20 to 30 hours of development and Fib which is labeled only after 20 to 30 hours. Fla is conserved, however, to the pluteus stage.The total acid-soluble protein content of chromatin remains constant to 22 hours of development. During the period of 22 to 45 hours, there is a slight loss of protein followed by a rapid loss from 45 to 70 hours such that at 70 hours only 20% remains.