Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Histones of terminally differentiated cells undergo continuous turnover

In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.     

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.     

Analysis of Histones Associated with Neuronal and Glial Nuclei Exhibiting Divergent DNA Repeat Lengths

Abstract: Total cerebral hemisphere nuclei purified from adult rabbit brain were subfractionated into neuronal and glial populations. Previous studies have shown that chromatin in neuronal nuclei is organized in an unusual nucleosome conformation compared with glial or kidney nuclei, i.e., a short DNA repeat length is present. We now analyze whether this difference in chromatin organization is associated with an alteration in the histone component of nucleosomes. Total histone isolated by acid/urea-protamine extraction of purified neuronal, glial, and kidney nuclei was analyzed by electrophoresis on SDS-polyacrylamide slab gels. Histone H1 that was selectively extracted from nuclei was also examined. Differences were not observed on SDS gels in the electrophoretic mobilities of histones associated with either the nucleosome core particle (histones H2A, H2B, H3, H4) or the nucleosome linker region (histone H1). Total histone and selectively extracted histone H1 were also analyzed on acid/urea slab gels that resolve histones on the basis of both molecular weight and charge differences. When analyzed in this system, differences with respect to electrophoretic mobility were not detected when comparing either selectively extracted histone H1 or total histone from neuronal and glial nuclei. Quantitative analyses were also performed and neuronal nuclei were found to contain less histone H1 per milligram DNA compared with glial or kidney nuclei. Neuronal nuclei also demonstrated a lower ratio of histone H1/core histone. These results suggest that the pronounced difference in chromatin organization in neuronal compared with glial nuclei, which is reflected by a short DNA repeat length in neurons, appears to be associated with quantitative differences in neuronal histone H1.     

H3 phosphorylation-dependent structural changes in chromatin. Implications for the role of very lysine-rich histones

Contrary to native H1/H5-containing chromatin where phosphorylation induces local structural changes affecting chromatin condensation, in stripped fibers phosphorylation of the totality of H3 molecules does not affect significantly chromatin conformation and DNA-protein interactions. Modification of H3 causes only a slight increase of flexibility of nucleosomal chains, despite important changes in histone topography revealed by immunochemical reactivity studies. We suggest that phosphorylation may only induce into the system the potential for dynamic change by modulating histone-histone interactions within and between nucleosomes, probably as a result of conformational change in the H3 protein. The signal for structural change would come from one or other factors (very lysine-rich histones, non-histones) that influence internucleosomal interactions at very specific locations in the chromatin, probably through protein-protein contacts. So, phosphorylation may modify a direct interaction between the N-terminal basic tail of H3 and very lysine-rich histones.     

Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones

Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120–140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.     

Duplicated nucleosome repeat generated in the erythrocyte chromatin by DNAse I. The role of lysine-rich histones

Dinucleosome periodicity of DNA fragmentation produced by DNAse I in nuclei of pigeon and trout erythrocytes differing in the content of histones H1 and H5 has been investigated. In spite of differences in the content of histone H5 (H1 to H5 ratio is approximately equal to 0.5 and 2 in pigeon and trout erythrocytes respectively) the double-nucleosome repeat was revealed clearly in pigeon and trout erythrocyte nuclei. To elucidate the role of lysine-rich histones we carried out the selective extraction of histone H1 from erythrocyte nuclei by a solution containing 0.3-0.35 M NaCl (pH 3.0) or cleavage of histones H1 and H5 by mild trypsinization in the presence of Mg2+ ions. It was shown that lysine-rich histones play a principal role in formation and maintenance of the so-called dinucleosomal chromatin structure.     

Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.     

Species- and cell-specific expression of H1 histones in tissue culture cells

The H1 histones of 25 tissue culture cell types of rat, mouse, human, and Chinese hamster origin were studied by ion-exchange chromatography. Fractionation of H1 histones revealed that each cell type expressed multiple H1 histone subfractions. The average number of four subfractions was the same as seen in tissues. The individual H1 histones exhibited species-specific chromatographic properties, and different cell lines from a given animal were capable of expressing cell-specific quantities of the H1 histone subtypes. Neither the species- nor cell-specific properties of the H1 histones could be abolished by alkaline phosphatase treatment, but such treatment did reduce the complexity of H1 histone subfractions in SV40-transformed cells. The direct comparison of rat liver and rat hepatoma cell (H-35) H1 histones by chromatographic and electrophoretic methods demonstrated that a cell line was capable of expressing the same variety of H1 histones as the tissue of origin. Variations in the content of H1 histone subtypes were seen in intraspecies comparisons in all four species. The variations were not reduced by recloning selected cells. The variations among subfractions were dramatic for Chinese hamster cell lines CHO, V79, and DON. Smaller variations could be seen in mouse cell lines and numerous genetic mutant human fibroblast cells and HeLa cells, but the latter did not appear to be specifically related to disease state. Since H1 histones link nucleosomes, the packing of nucleosomes in the chromatin of tissue culture cells has the same potential variability as in tissues.     

Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.

UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.     

Accessibility of histone oligomers to the action of trypsin in a solution or in chromatin with different degrees of compactness

The accessibility to trypsin of "core" histones within the dimer (H2A-H2B), tetramer (H3-H4)2, octamer (H2A-H2B-H3-H4)2 and in chromatin was studied. It was shown that the hydrolysis of histones H2A and H2B within the dimer and octamer occurs in essentially the same way. The tetramer (H2-H4)2 becomes more compact with an increase in the ionic strength. Some of the tetramer (H3-H4)2 sites within the octamer are protected against trypsin. It was demonstrated that in terms of the histone accessibility to trypsin chromatin can exist in three states, i.e., tightly packed (in the presence of histone H1 and bivalent cations), intermediate (in the absence of histone H1 or bivalent cations) and folded (in the absence of histone H1 and bivalent cations). The folding of histones in neither of these chromatin states coincides with that within the octamer in 2M NaCl.     

NUCLEAR HISTONE PROTEINS OF GAMETES IN AN OOGAMOUS AND TWO ISOGAMOUS BROWN ALGAE1

Nuclear basic proteins (histones) were studied in male and female gametes of the isogamous brown algae, Colpomenia bullosa (Saunders) Yamada and Analipus japonicus (Harvey) Wynne and sperm of the oogamous Cystoseira hakodatensis (Yendo) Fensholt by using SDS‐ and AUT‐PAGE. Four major core histones and several linker histone H1s were detected by electrophoresis. Each of the core histones was identified by amino acid sequence analysis and peptide mapping. Electrophoresis patterns of histones were the same in male and female gametes and quite similar between the two species. The composition of histone H1s in conspicuously condensed sperm nuclei of C. hakodatensis was different from that in isogamous gametes. Electrophoresis after micrococcal nuclease digestion of chromatin in male and female gamete nuclei of C. bullosa and A. japonicus and sperm of C. hakodatensis resulted in regular ladder patterns of DNA fragments (ca. 200 base pair). The chromatin of the brown algal gametes thus has the typical nucleosome structure. These results showed that chromatin condensation in sperm nuclei of C. hakodatensis was associated with a modification of linker histone H1 but not by change of core histones, replacement by other basic proteins, changes of repeating patterns, or disappearance of nucleosomes.     

Chemical crosslinking of histone H1° to histone neighbours in nuclei and chromatin

Crosslinking of histones in mouse liver nuclei and extended chromatin with a bifunctional reagent leads to the formation of H1H1° heterodimers as well as H1°H1° homodimers. H1° can be also crosslinked to the core histones. Thus, the location of histone H1° within the basic repeating chromatin structure seems to be analogous to that of H1 histone.     

Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase.

At concentrations normally used to inhibit eukaryotic type II topoisomerase activity (100-1000 micrograms/ml) novobiocin binds core histones. Approximately 15 moles of novobiocin bind per mole of histone resulting in histone precipitation from solution in either 0.15 M or 2 M NaCl. The interaction between novobiocin and proteins appears to involve arginine residues: histones H3 and H4 (13.5 and 14 mole percent arginine) are precipitated at lower novobiocin concentrations than histones H2A and H2B (9.5 and 6.5 mole percent arginine). Furthermore, polyarginine but not polyornithine competes for novobiocin in histone precipitation. Moreover, histones with arginine residues modified with 1,2-cyclohexanedione are soluble in 1000 micrograms/ml novobiocin. Because novobiocin can remove histones from solution as well as inhibit topoisomerase activity, and because both of these events can alter DNA topology, novobiocin should be used with caution in experiments designed to implicate topoisomerase activity in chromatin dynamics.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.