Large scale preparation of nucleosomes containing site-specifically chemically modified histones lacking the core histone tail domains

The core histone tail domains are critical regulators of chromatin structure and function and modifications such as acetylation of lysine residues within the tails are central to this regulation. Studies have shown that the removal of core histone tail domains by trypsinization in which one-half to two-thirds of each core histone tail domain are removed in gross aspects mimics the acetylation of core histone tails. In addition, removal of the tails has been useful in understanding general tail function. Thus, removal of native core histone tails by trypsinization is a widely used method. In addition, many in vitro studies now employ core histones site-specifically modified with photo activatable cross-linking probes or fluorescent probes. However, in our experience, standard methods employing trypsinized donor chromatin for reconstitution of nucleosomes containing certain chemically modified histones lacking the core histone tail domains are not uniformly applicable. Here, we describe various methods for preparing nucleosomes containing a core histone modified with a cross-linking agent, APB, and lacking the core histone tail domains.     

Binding of non-histone chromosomal protein HMG1 to histone H3 in nucleosomes detected by photochemical cross-linking

The interaction of non-histone chromosomal protein HMG1 with core histones in nucleosomes was studied via reconstitution and photochemical cross-linking. The results obtained indicated that photoaffinity-labeled HMG1 interacted in nucleosomes with histone H3. Similar experiments with peptides derived from HMG1 by V8 protease digestion allowed to identify N-terminal domain of HMG1 (peptide V3) as a binding region for histone H3 in nucleosomes.     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Nucleosomal structure as probed by H3 histone thiol reactivity

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available toN-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.     

Nucleosomal structure as probed by H3 histone thiol reactivity. Conformation of H3 histone variants is differently affected by thiol group reagents

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available to N-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.     

H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro

Nucleosome formation has been studied in a system containing relaxed Col E1 DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assembled in the presence of the H1 histone, polylysine (Mr 20 000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.     

cis- and trans-diamminedichloroplatinum(II) interstrand cross-linking of a defined sequence nucleosomal core particle

Interstrand cross-linking studies with the antitumor drug cis-diamminedichloroplatinum(II) and its clinically inactive isomer, trans-diamminedichloroplatinum(II), were performed on a fragment of the 5S rRNA gene of Xenopus borealis in the free and nucleosomal state. 5S nucleosomes were formed via histone octamer exchange from chicken erythrocyte core particles. Native polyacrylamide gel electrophoresis was used to probe the ability of platinated DNA to reconstitute into core particles. Both isomers negatively impacted reconstitution when histones were present during incubation with the drug. When histones were not present during the drug treatment, platinated DNA was successfully reconstituted into core particles. These results suggest that platination of histones impedes reconstitution of free DNA. However, already-formed core particles were not disrupted upon platination. Sites of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. We found both site-specific enhancement and depression of cis-diamminedichloroplatinum(II) cross-linking in the nucleosomal samples relative to free DNA at both drug concentrations that were tested (0.01 and 0.0025 mM). trans-Diamminedichloroplatinum(II) exhibited no detectable differences in the interstrand cross-linking of free and nucleosomal samples.     

Studies on protein organization of nucleosomes using cross-linking

When chromosomal proteins in chromatin or in mononucleosomes were extensively cross-linked with an imido ester, the H1-containing nonameric histone complex was revealed. In this complex, histone H1 is connected with the octamer of core histones. The cross-linking of H1 to the octamer is realized preferentially through H2a and H3 histones. Some HMG (high mobility group) proteins located presumably in the linker regions of a nucleosome fiber also take part in the formation of dimers, possibly with the histones of a nucleosomal core. The results suggest mutual interactions between some linker-associated proteins and intranucleosomal histones. Experiments involving extensive cross-linking of proteins in the purified mononucleosome subfractions demonstrated differences in the organization of core histones between complete nucleosomes and nucleosomes lacking H1.Abbreviations HMG proteins high mobility group proteins - DMA dimethyladipimidate dihydrochloride - DMP dimethyl-3,3-dithio-bis-propionimidate dihydrochloride     

DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.     

Interactions between core histones and chromatin at physiological ionic strength

Addition of core histones to chromatin or chromatin core particles at physiological ionic strength results in soluble nucleohistone complexes when polyglutamic acid is included in the sample. The interaction between nucleosomes and added core histones is strong enough to inhibit nucleosome formation on a closed circular DNA in the same solution. Complexes consisting of core particles and core histones run as discrete nucleoprotein particles on polyacrylamide gels. Consistent with the electrophoretic properties of these particles, protein cross-linking with dimethyl suberimidate indicates that added core histones are bound as excess octamers. Histones in the excess octamers do not exchange with nucleosomal core histones at an ionic strength of 0.1 M and can be selectively removed from core particles by incubating the complexes in a solution containing sufficient DNA. Under conditions where added histones are confined to the surface of chromatin, the excess histones are mobile and can migrate onto a contiguous extension of naked DNA and form nucleosomes.     

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.     

ATP dependent histone phosphorylation and nucleosome assembly in a human cell free extract.

Physiologically spaced nucleosome formation in HeLa cell extracts is ATP dependent. ATP hydrolysis is required for chromatin assembly on both linear and covalently closed circular DNA. The link between the phosphorylation state of histones and nucleosome formation has been examined and we demonstrate that in the absence of histone phosphorylation no stable and regularly spaced nucleosomes are formed. Phosphorylated H3 stabilizes the nucleosome core; while phosphorylation of histone H2a is necessary to increase the linker length between nucleosomes from 0 to approximately 45 bp. Histone H1 alone, whether phosphorylated or unphosphorylated, does not increase the nucleosome repeat length in the absence of core histone phosphorylation. Phosphorylations of H1 and H3 correlate with condensation of chromatin. Maximum ATP hydrolysis which is necessary to increase the periodicity of nucleosomes from approximately 150 to approximately 185 bp, not only inhibits H1 and H3 phosphorylation but facilitates their dephosphorylation.     

Unfolding of nucleosome cores induced by chemical acetylation of histones

Relative accessibility of nucleosomal histones to acetic anhydride during acetylation has been studied as a function of concentration, pH and ionic strength of the solution using high-resolution gel-electrophoresis. It was shown that about 80% of lysine residues in nucleosomal histones and 100% of the same residues in histone complexes without DNA in 2 M NaCl are accessible to the modification, which is proved by the localization of the majority of lysine residues in nucleosomes near the surface of the histone octamer, by their participation in ionic interactions with DNA and, probably, in histone-histone contacts. Gel-electrophoretic experiments with nucleosomes and studies of the histone resistance to mild trypsinolysis indicated that neither nucleosomes themselves nor histone octamers are affected even though 50% of lysine residues in histones have been acetylated. The process of acetylation is accompanied by the growing tendency of histones to participate in mild trypsinolysis and by a gradual decline in electrophoretic mobility and in the value of the sedimentation constant. The circular dichroism spectra and the microscopic appearance of nucleosomes are also markedly changed. These results suggest that a gradual unfolding of nucleosomes occurs when 5 or more lysine residues in the nucleosomal histones have been acetylated.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.