Studies on the role of histones H1 (f1) and H5 (f2c) in chromatin structure

Chicken erythrocyte and liver nuclei, isolated and fixed in isotonic saline, contained compact chromatin fibers about 200 Å in diameter. Fibers very similar in dimension and appearance are usually visible in thin sections of fixed nuclei in situ and probably represent chromatin organization close to the native state. After suspension of isolated nuclei in a mildly hypotonic buffer, chromatin fibers extended, became reduced in diameter and apparently unraveled in places. Under such conditions, new detail was revealed suggestive of both helical structure and of subunit organization. The fibers extended and contracted reversibly, changing in diameter from 200 Å to about 100 Å when alternately exposed to isotonic saline and to distilled water. The 200 Å fibers were irreversibly lost, however, following extraction of nuclei with high concentrations of NaCl which selectively removed H1 from liver and H1 plus H5 from erythrocyte chromatin. After extraction of more tightly complexed histones, the residual chromatin consisted mainly of fine filaments less than 30 Å thick. These results suggest a model for native chromatin fibers in which sub-unit organization and coiled configuration are combined, and in which histones H1 and H5 play an integral role in the maintenance of structure.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

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.     

Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid-phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.     

Regulation of the higher-order structure of chromatin by histones H1 and H5

Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously. The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA. The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes. The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests. The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.     

Relaxation of chromatin structure upon removal of histones H2A and H2B

Modification of chromatin from chicken erythrocytes with dimethylmaleic anhydride is accompanied by its solubilization and the dissociation of histones H1, H5, H2A and H2B. Histone H1 is the first to dissociate and H5 the last. After regeneration of the modified amino groups, residual chromatin preparations with different histone composition were studied by circular dichroism and thermal denaturation. In addition to the effects produced by the lack of histones H1 and H5, both techniques show a substantial relaxation of chromatin structure induced by the loss of histones H2A and H2B, which appear to play an important role in the superhelical folding of DNA.     

Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.     

Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.     

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.     

DNA repeat lengths of erythrocyte chromatins differing in content of histones H1 and H5

Among the erythrocytes of chicken, trout, carp, and sucker, the relative proportion of the lysine-rich histone H5 varied from 20 to 0% of the total histones. Following digestion of nuclear chromatin with micrococcal nuclease, each of them displayed a longer DNA repeat length and greater repeat length heterogeneity than found in liver chromatin. Fish erythrocytes possessed similar repeat lengths of 207-209 base pairs which was 10-12 base pairs shorter than in chicken erythrocyte chromatin and approximately 10 base pairs longer than in liver chromatin. No correlation existed between the DNA repeat length or repeat length heterogeneity and the relative proportion of H5.     

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.     

Partial amino acid sequence and functional aspects of histone H1 proteins in Trypanosoma brucei brucei

Summary— Trypanosoma brucei brucei, a protozoan parasite of wild and domestic animals in Africa, is related to the pathogenic agent of human sleeping sickness. Four H1 histone proteins were isolated from nuclei of procyclic culture forms and cleaved with proteases. Amino acid sequence analysis of purified fragments indicated the presence of variants which displayed sequence identities as compared to the C-terminal domain of human H1. Substitutions of amino acids and posttranslational modifications of the histones in iT b brucei H1 may influence protein conformation and histone-histone as well as histone-DNA interactions in the chromatin of the parasite. Digestion of soluble chromatin with immobilized trypsin at low and high ionic strengths indicated an internal localization of H1 in the condensed chromatin. The influence of histone H1 of T b brucei on the compaction pattern of the chromatin was investigated by dissociation and reconstitution experiments. Electron microscopy revealed that trypanosome H1 was able to induce condensation of the chromatin of the parasite and of rat liver into dense tangles. After dephosphorylation of H1, 30 nm fibers were induced in rat liver chromatin, while the resulting fibers were distinctly thinner in T b brucei. It can be concluded that the absence of 30 nm fibers in T b brucei chromatin cannot be explained by the divergent variants and posttranslational phosphorylations of H1 only but rather by the influence of both, the divergent core histones, previously described, and H1 properties.     

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.     

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.     

Differential response of avian red blood cell nucleosomes to heparin

In avian erythrocyte chromatin, heparin interacts differentially with H1, H5 and the nucleosomal core histones. In non-erythroid cells, a partial extraction of H2A, H2B and H1 yields H3/H4/DNA complexes and particles of unchanged nucleosomal composition. The assay system for this heparin effect includes sucrose gradients, formaldehyde fixation and cesium chloride gradient centrifugation. A comparison of avian erythrocyte nucleosomes with chromatin subunits from precursor cells shows that H5 interferes with the heparin effect whereas a removal of H5 renders the core histones accessible to the polyanion.     

Chromatin organization and basic nuclear proteins in the male germ cells of Rana tigerina

The process of chromatin condensation during spermiogenesis in Rana tigerina is similar to the heterochromatization in somatic cells, where 30 nm fibers are coalesced together into a dense mass in spermatozoa without changing their initial size and nucleosomal organization. This conclusion was supported by the finding that the full set of core histones (H2A, H2B, H3, H4) are still present in sperm chromatin, but histone H1 is replaced by its variant, H1V. Rabbit anti-sera were raised against histone H3, H1, H1V, and H5 (H1 variant in chick erythrocyte). Anti-histone H1 antiserum cross-reacted with histone H1V, which implied the presence of a common epitope. Anti-histone H1V and H5 also showed cross-reaction with each other but not with histone H1, which implied the presence of a common epitope not shared by histone H1. Immunocytochemical studies, using the above antibodies as probes, showed that histones H3 is present in all steps of spermatogenic and spermiogenic cells, and somatic cells including red blood cells, Sertoli cells, and Leydig cells, while histone H1 is present in all of the cells mentioned except in spermatozoa where it is replaced by histone H1V. Histone H1V appears in the early spermatids starting from spermatid 1 (St1), and it persists throughout the course of spermatid differentiation into spermatozoa. Histone H1V is also found in chromosomes of metaphase spermatocyte and red blood cells. Thus histone H1V may cause the final and complete condensation of chromatin in Rana spermatozoa, a process which is similar to the heterochromatization occurring in somatic cells such as metaphase chromosome and chick erythrocyte nucleus.