Differential association of linker histones H1 and H5 with telomeric nucleosomes in chicken erythrocytes.

Rat liver telomeric DNA is organised into nucleosomes characterised by a shorter and more homogeneous average nucleosomal repeat than bulk chromatin as shown by Makarov et al. (1). The latter authors were unable to detect the association of any linker histone with the telomeric DNA. We have confirmed these observations but show that in sharp contrast chicken erythrocyte telomeric DNA is organised into nucleosomes whose spacing length and heterogeneity are indistinguishable from those of bulk chromatin. We further show that chicken erythrocyte telomeric chromatin contains chromatosomes which are preferentially associated with histone H1 relative to histone H5. This contrasts with bulk chromatin where histone H5 is the more abundant species. This observation strongly suggests that telomeric DNA condensed into nucleosome core particles has a higher affinity for H1 than H5. We discuss the origin of the discrimination of the lysine rich histones in terms of DNA sequence preferences, telomere nucleosome preferences and particular constraints of the higher order chromatin structure of telomeres.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Higher order structure in a short repeat length chromatin

Polynucleosomes from calf brain cortical neurone nuclei have an average repeat length of less than 168 base pairs. The ability of this material to adopt higher order structure has been assessed by various physical techniques. Although containing on average less DNA per nucleosome than is required to form a chromatosome, this short repeat length chromatin folded in an H1 dependent manner to a structure with properties similar to those observed for longer repeat length chromatins such as that of chicken erythrocyte (McGhee, J.D., D.C. Rau, E. Charney, and G. Felsenfeld, 1980, Cell, 22:87-96). These observations are discussed in the context of H1 location in the higher order chromatin fiber.     

Modulation of histone H5-nucleosome interactions by H5 phosphorylation

In nucleosomal particles of 180 base pairs, part of the histone H5 binding site is preserved. After fluorescein labelling of H5 from chicken erythrocytes comparative equilibrium binding studies have been performed and on these particle as well as on core particles (140 base pairs) and on free DNA (180 base pairs). While nucleosomal particles can accommodate about the same number of H5 molecules as the free DNA derived from it, affinities are decreased by a factor of 3. A further decrease by factors of 3–4 is the consequence of phosphorylating three of the H5 serines: hence phosphorylation should facilitate thermodynamically controlled complexing of red cell chromatin during erythropoiesis. The most dramatic effect of an H5 phosphorylation is a reduction in the binding sites from 56 to 36 nucleotides (free DNA) and an even more pronounced effect upon interacting with nucleosomes which should make the H5-chromatin association sterically favourable. Related studies with protamines from herring are included for comparison.     

The subunit structure of chromatin from Physarum polycephalum.

Nucleosome DNA repeat lengths in Physarum chromatin, determined by nuclease digestion experiments, are shorter than those observed in most mammalian chromatin and longer than those reported for chromatin of certain other lower eukaryotes. After digestion with staphylococcal nuclease for short periods of time an average repeat length of 190 base pairs is measured. After more extensive digestion an average repeat length of 172 base pairs is measured. Upon prolonged digestion DNA is degraded to an average monomer subunit length of 160 base pairs, with only a small amount of DNA found in lengths of 130 base pairs or smaller. Mathematical analysis of the data suggests that the Physarum nucleosome DNA repeat comprises a protected DNA segment of about 159 base pairs with a nuclease-accessible interconnecting segment which ranges from 13 to 31 base pairs. The spacing data are compatible with measurements from electron micrographs of Physarum chromatin.     

Influence of histone H5 on mononucleosome structure during differentiation in the avian erythroid series

Changes in nucleosome repeat length during avian erythroid development have been previously correlated with changes in H5 content. In order to determine the effects of H5 on the length of DNA in mononucleosomal particles as a function of differentiation, a two-dimensional electrophoretic system was used to analyse DNA and histones of particles generated by micrococcal nuclease digestion of nuclei from several stages of erythroid development. Although the relative proportions of H5- to H1-containing mononucleosomes increased during development, only in mature erythrocytes did H5 protect a greater length of linker DNA from micrococcal nuclease digestion than did H1. These results suggest that changes in average nucleosome repeat length during erythroid development can be attributed only partially to an increase in the proportion of H5-containing nucleosomes which contribute to this average.     

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.     

Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.     

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.     

Preparation and properties of a nucleosomal particle containing 180 base pairs of DNA

If chromatin from chicken erythrocytes is enzymatically degraded in the presence of histone H5, nucleosomal DNA usually exceeds the size of 140 base pairs. Conditions are derived allowing the isolation of a 180 base pair particle which is subsequently characterized by histone binding and thermal denaturation studies. Association of H5 to such a particle is cooperative and occurs with a larger affinity than binding to specimens in the range of 140– 170 base pairs. Thermal denaduration studies show that some of the extra DNA participates in the main transition at 74°C (1 mm Na-cacodylate) indicating a tight binding to the histone core. Another part of the extra segment is loosely associated with the core but also distinct from free DNA.     

Protection of discrete DNA fragments by the complex H1-octamerhistones or H5-octamerhistones after micrococcal nuclease digestion.

Several authors, including ourselves, have reported the existence of chromatosomes with DNA size larger than 166 bp in bird erythrocyte chromatin. It was tempting to correlate this increased DNA size with the presence of histone H5. In order to substantiate this hypothesis, we performed a micrococcal nuclease digestion kinetic on: chicken erythrocyte chromatin, either native, selectively depleted from H1, or from H1 and H5; and rat liver chromatin, either native or partially H1 depleted. The comparative analysis of the lengths of DNA in the chromatosome size region led to the following conclusions: - denaturing gels clearly reveal a first discrete pause at 178 nucleotides in H1 depleted chicken erythrocyte chromatin as well as in partially H1-depleted rat liver chromatin, before the material accumulates at the next intermediate 166 nucleotide chromatosome pause. - the generation of all discrete chromatosome bands is critically dependent on low ionic strength conditions and low Ca++ concentrations during the digestion, suggesting it may result from the protection of DNA cleavage sites by histone H5 or H1, C or N terminal domains.     

Quantitative analysis of the digestion of yeast chromatin by staphylococcal nuclease.

The DNA in intranuclear yeast chromatin is protected from rapid staphylococcal nuclease degradation so as to yield an oligomeric series of DNA sizes. The course of production and disappearance of the various oligomers agrees quantitatively with a theory of random cleavage by the enzyme at uniformly susceptible sites. The sizes of the oligomers are integral repeats of a basic size, about 160 base pairs, and 80-90% of the yeast genome is involved in this repeating structure. Within this repeat there exists a 140 base pair core of more nuclease-resistant DNA. During the course of digestion, the sizes of the oligomers decrease continuously. The widths of the distribution of DNA sizes increase in order: monomer (1 X repeat size, half width = 5-7 base pairs) less than dimer (2 X repeat size, half width = 30 base pairs) less than trimer (3 X repeat size, half width = 40-45 base pairs). The yeast genome thus seems to have variable spacing of the nucleaseresistant cores, to produce the average repeat size of about 160 base pairs. Also, the presence of more than one species of monomer and dimer at certain times of digestion suggests a possible heterogeneity in the subunit structure.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

A model chromatin assembly system. Factors affecting nucleosome spacing

Poly[d(A-T)].poly[d(A-T)], when reconstituted with chicken erythrocyte core histones and subsequently incubated with sufficient histone H5 in a solution containing polyglutamic acid, forms structures resembling chromatin. H5 induces nucleosome alignment in about two hours at physiological ionic strength and 37 degrees C. The nucleosome spacing and apparent linker heterogeneity in the assembled nucleoprotein are very similar to those in chicken erythrocyte chromatin. Also, condensed chromatin-like fibers on the polynucleotide can be visualized. The binding of one mole of H5 per mole of core octamer is necessary to generate the physiological nucleosome spacing, which remains constant with the addition of more H5. The nucleosome repeat length is not a function of the core histone to poly[d(A-T)] ratio for values lower than the physiological ratio. With increasing ratios, in excess of the physiological value, nucleosome spacing first becomes non-uniform, and then takes on the close packing limit of approximately 165 base-pairs. In addition to eliminating possible base sequence effects on nucleosome positioning, poly[d(A-T)] allows nucleosomes to slide more readily than does DNA, thereby facilitating alignment. Evidence is presented that polyglutamic acid facilitates the nucleosome spacing activity of histone H5, primarily by keeping the nucleoprotein soluble. This model system should be useful for understanding how different repeat lengths arise in chromatin.     

Introns of the chicken ovalbumin gene promote nucleosome alignment in vitro.

A defined in vitro chromatin assembly system was used to examine the nucleosome alignment induced by histone H5 throughout a 12 kilobase pair chicken genomic DNA fragment containing the ovalbumin gene. In contrast with total fragmented chicken DNA and several anonymous cloned fragments, much of the gene permitted histone H5 to space nucleosomes at physiological intervals in an extended array. Nucleosomes at the 3'-end of the gene and on approximately 4 kilobase pairs of 5'-flanking ovalbumin sequence did not become aligned to appreciable extents. Analysis of cloned 2-3 kilobase pair subfragments suggested that a strong nucleosome alignment signal, specifying a 196 +/- 5 base pair repeat exists in intron E. A second discrete region of the gene, which mapped approximately to intron A, exhibited nucleosome alignment with a spacing periodicity of about 200 base pairs. The ovalbumin cDNA did not permit nucleosome alignment. These findings suggest that some of the introns contain signals that direct nucleosome alignment over the ovalbumin gene in a way conducive to its regulation.     

Nucleosome structure in Aspergillus nidulans

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

Binding of androgen receptor to prostatic chromatin requires intact linker DNA.

Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.     

Chicken erythrocyte chromatin and nuclear envelope antigens

Chromatin and inner layer nuclear envelope were isolated from chicken erythrocyte nuclei. Two antisera against dehistonized chromatin and nuclear envelope of chicken erythrocytes were obtained. Using the antiserum against dehistonized chromatin of erythrocytes we found: the presence of the antigens at approximate mol. wts of 56,000 and 77,000 tightly bound with DNA and characteristic of only erythrocyte chromatin; localized antigens at approximate mol. wts of 63,000, 68,000 and 92,000 tightly bound with DNA and common only for chromatin and nuclear envelope of chicken erythrocytes; heterogeneity of the antigens tightly bound with DNA. Using the antiserum against inner layer nuclear envelope we did not find antigens specific only for nuclear envelope and absent in erythrocyte chromatin. Some of the antigens were present in the control preparations of chicken liver chromatin and may be regarded as being species specific.     

Structure of transcriptionally-active chromatin subunits.

Rat liver chromatin is organized into regions of DNA which differ in degree of susceptibility to attack by the endonucleases DNase I and DNase II. The most nuclease-sensitive portion of chromatin DNA is enriched in transcribed sequences. This fraction may be separated from the bulk of chromatin by virtue of its solubility in solutions containing 2 mM MgCl2. Both transcribed and nontranscribed regions of chromatin are organized into repeating units of DNA and histone, which appear as 100 A beads in the electron microscope. The length of DNA in the repeat unit is the same for these two classes of chromatin (198 +/- 6 base pairs in rat liver); however, the subunits of active, Mg++-soluble chromatin differ from the nucleosomes of inactive regions of chromatin in several respects. Active subunits are enriched in nascent RNA and nonhistone protein and exhibit higher sedimentation values than the corresponding subunits of inactive chromatin.