Proximity and accessibility studies of histones in nuclei and free nucleosomes.

Histone proximity in chromatin was studied with the cleavable crosslinking reagent, dithiobissuccinimidyl propionate. Crosslinks between H4 and H2a, H4 and H2b, H4 and H3, H2a and H2b, H2b and H3 were found. H1 is also crosslinked to the nucleosomal histones. In nuclei, unsheared chromatin, and H1 depleted chromatin, the four nucleosomal histones are crosslinked at similar relative rates both in 5 mM salt and 100 mM salt. After micrococcal nuclease treatment to generate nucleosomes, H2a and H2b are crosslinked faster than H4 and H3. C14-NEM titration of thiopropionate residues bound to each histone shows that H2a and H2b are more accessible to this reagent after nuclease treatment but that the increased binding was not sufficient by itself to explain the increase in crosslinking. Bolton Hunter reagent was used to further study the accessibility of the four nucleosomal histones in whole chromatin and nuclease digested chromatin. These studies showed that salt increases the accessibility of all four histones while nuclease treatment decreases H4 accessibility.     

DNA folding by histones: the kinetics of chromatin core particle reassembly and the interaction of nucleosomes with histones.

The kinetics of the chromatin core particle reassembly reaction in solution were quantitatively studied under conditions such that nucleohistone aggregation did not occur. Core particles, salt-jumped rapidly by dilution from 2.5 m-NaCl (in which DNA and histones do not interact) to 0.6 m-NaCl (in which core particles are nearly intact), reassemble in two distinct time ranges. Approximately 75% of the DNA refolds into core particle-like structures “instantaneously” as measured by several physical and chemical techniques with dead times in the seconds to minutes time range. The remaining DNA refolds with relaxation times ranging from 250 minutes at 0 °C to 80 minutes at 37 °C; this slow effect cannot be attributed to sample heterogeneity. The fraction of slowly refolding DNA and the slow relaxation time are independent of the core particle concentration. Transient intermediates present during the slow phase of refolding were identified as free DNA and core particle-like structures containing excess histone. Mixing experiments with DNA, histones, and core particles showed that core particle-histone interactions are responsible for the slow kinetics of DNA refolding. Upon treatment of reassembling core particles with the protein crosslinking reagent, dimethylsuberimidate, the slow phase of the reassembly reaction was arrested and a 13 S particle containing DNA and two octamers of histone was isolated. Consistent with the nature of this kinetic intermediate, it is shown that in 0.6 m-NaCl, core particles co-operatively bind at least one additional equivalent of histones with high affinity in the form of excess octamers. Also, core particles continue to adsorb considerably more histones with a weaker association constant of the order 10⁵m^?1 (in units of octamers) to a maximum value of 12 ± 2 equivalents (octamers) per core particle. The sedimentation coefficient increases with the two-thirds power of the molecular weight of the complex, as it would in the case of clustered spheres.A reassembly mechanism consistent with the data is presented, and other simple mechanisms are excluded. In the proposed mechanism, core particles reassemble very rapidly and compete effectively with DNA for histones such that approximately one-third of the particles initially formed are complexed with an excess octamer of histones, and 25% of the total DNA remains uncomplexed. The amount of this unusual reaction intermediate decays slowly to an equilibrium value of about 10%, thereby leaving 9% of the total DNA uncomplexed. Approximate values are calculated for the free energies, rate constants, and two of the activation energies which characterize this migrating octamer mechanism. This mechanism provides a means whereby histone octamers can be temporarily stripped off DNA at a modest free energy cost, approximately 2.6 kcal per nucleosome. Also, the properties of excess histone adsorption by chromatin and octamer migration suggest an efficient mechanism, consistent with observations by others, for nucleosome assembly in vivo during replication.     

HAMLET interacts with histones and chromatin in tumor cell nuclei

HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.     

Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.     

A site of discontinuity in the interaction between DNA and histones in nucleosomes of sea urchin embryo chromatin.

Digestion of chromatin DNA in nuclei of sea urchin embryos with pancreatic nuclease and with micrococcal nuclease give additional details concerning the interaction between DNA and histones. A specific site of hydrolysis appears to be located on the nucleosome in such a position as to split the DNA unit length in two equivalent fragments of about 60–70 base pairs in length. The complete digestion of chromatin DNA appears to depend on the low stability of the nucleosome containing the split DNA fragments.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Comparison of the primary structure of reconstructed minimal nucleosomes and nucleosomes isolated from the chromatin

We have reconstructed nucleosomes from a histone octamer (H2A, H2B, H3, H4)2 and DNA 146 b.p. or 2-3 thousands b.p. in length. Comparison by means of DNA-histone cross-links of the primary organization of minimal nucleosomes obtained by reconstruction or isolated from chromatin of chicken erythrocyte nuclei has demonstrated a high similarity in histone location on their DNAs. Simultaneously, there have been observed some variations in the character of interaction for all core histones with DNA on nucleosomes. Thus, the cross-link of histone H4 with DNA of a core particle at H4 sites (65), unlike H4(55) and H4(88) sites, significantly depends on the superstructure of chromatin, ionic strength of solution and the presence of denaturating agents. All these differences are expected to probe the existence of conformational isomers for core particles. (Bracketed is the distance from the histone interaction site with the DNA of the core particle to the DNA 5'-terminus.)     

The role of histone H1 and non-structured domains of core histones in maintaining the orientation of nucleosomes within the chromatin fiber

Calf thymus chromatin was digested with trypsin and the structural alterations which occurred were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.     

Deviant nucleosomes: the functional specialization of chromatin

Regulatory proteins exist with strong sequence and structural similarities to the bistone proteins. Molecular genetic and cell biological analyses suggest that these proteins are localized at particular sites within the chromosome. Their assembly into nucleosomal structures confers specialized functions to individual chromosomal domains.     

The binding of histones H1 and H5 to chromatin in chicken erythrocyte nuclei.

The binding curves of histones H1 and H5 to chromatin in nuclei have been determined by a novel method which utilises the differential properties of free and bound histones on cross-linking with formaldehyde. The dissociation is thermodynamically reversible as a function of [NaCl]. The binding curves are independent of temperature over the range 4 degrees - 37 degrees C and independent of pH over the range 5.0 to 9.0. The curves are sigmoid, indicating co-operative dissociation with NaCl. The standard free energy of dissociation in 1 M NaCl for H1 is 0.5 Kcals/mole and for H5 is 3.5 Kcals/mole.     

Metabolism of histones and non-histone proteins in liver cell nuclei and chromatin from rats of various ages

The metabolism of various classes of histones and nonhistone proteins in intact nuclei and in liver chromatin of albino Wistar rats aged 1, 3, 12 and 24 months, was studied. It was shown that in the course of postnatal development the metabolism of nonhistone proteins extracted with 0.14 M NaCl in murine liver is increased. Later in ontogenesis, the incorporation of labeled precursors into proteins HMG 14 and HMG 17 decreases; the specific radioactivity of proteins HMG 1 + 2 is higher in 3- and 24-month-old animals. The intensity of metabolism of nonhistone proteins and histones is higher within the composition of the chromatin complex than in the intact nucleus at all stages of postnatal development. Among other histone proteins, histones H1 are characterized by the highest level specific radioactivity in rats of all age groups.     

Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands

Density labeling procedures have been utilized to study the dynamics of histone-histone interactions in vivo. Cells were labeled for 60 min with dense amino acids, and the label was chased for up to 22 h (two replication events for these cells). Nuclei were isolated and treated with formaldehyde to stabilize the histone-histone interactions with a covalent cross-link that produces an octameric complex of two each of H3, H2B, H2A, and H4. This complex was then extracted from the DNA and analyzed on density gradients. The results indicate that new H3,H4 deposits as a tetramer and does not dissociate in the subsequent chases. New H2A,H2B deposited as a dimer and also does not dissociate in subsequent chases. These new histones form hybrid octamers with old histones. On the basis of the new:old ratio in the hybrid octamers, we propose that additional old H2A,H2B from elsewhere in the genome interacts with tetramers of new H3,H4 to form the newly synthesized nucleosomes. It is also observed that 5% of the cross-linked complexes produced by formaldehyde are octamer-octamer (dioctamer). Upon analysis of the density of the dioctamer, the hybrid octamers were found adjacent to octamers that were homogeneous with respect to containing normal density histones. Control experiments are presented to demonstrate that the octamer-octamer cross-links are a product of intrastrand and not interstrand interactions between nucleosomes. These same control experiments also indicate that these procedures do not induce histone exchange during the preparative procedure prior to density gradient analysis. The significance of these results with regard to the dynamics of histone-histone interactions at the replication fork and the potential role in the maintenance of differentiation is discussed.     

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.     

Formation of hybrid nucleosomes cantaining new and old histones.

5 mM hydroxyurea (HU) inhibits DNA synthesis in mouse P815 cells by 94-97% in less than 1 hr. Nevertheless, histone synthesis continues and newly-synthesised histones are incorporated into non-replicating chromatin at a rate of about 20% of that in control exponentially-growing cells. To study the organization of these histones in chromatin P815 cells were treated with 5 mM HU in medium containing dense (15N, 13C, 2H) - substituted amino acids. After inhibition of DNA synthesis, newly-synthesised histones were labelled with (3H)-arginine. The cells were harvested 90 min later, and mono- and oligonucleosomes were prepared and analysed on metrizamide-triethanolamine (MA-TEA density gradients. Analysis of the distribution of 3H-labelled histones in these gradients shows that they are incorporated into hybrid mononucleosomes containing both new and old histones. It is also shown that these hybrid nucleosomes are not randomly distributed, but show a certain tendency to be clustered in certain chromatin regions.     

Positioning of nucleosomes reconstituted with xeroderma pigmentosum and normal histones

Positioning of nucleosomes was examined in a reconstituted system using a plasmid DNA and histones from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells. The present studies indicate that the arrangement of nucleosomes, composed of normal human histones, in a region near the SV40 origin of replication on the plasmid DNA, is nonrandom. The alignment of nucleosomes in this region was not affected by the presence of histone H1. No difference in nucleosome positioning was observed when the nucleosomes were composed of histones from XPA cells.     

Proteolytic processing of h1-like histones in chromatin: a physiologically and developmentally regulated event in Tetrahymena micronuclei

Micronuclei isolated from growing cells of Tetrahymena thermophila contain three H1-like polypeptides alpha, beta, and gamma. Micronuclei isolated from young conjugating cells (3-7 h) also contain a larger molecular weight polypeptide, X, which is being actively synthesized and deposited into these nuclei (Allis, C. D., and J. C. Wiggins, 1984, Dev. Biol., 101:282-294). Pulse-chase experiments (with growing and conjugating cells) suggested that X is a precursor to alpha and that alpha is further processed to gamma and a previously undescribed and relatively minor species, delta. These precursor-product relationships were supported by cross-reactivity with polyclonal antibodies raised against alpha and peptide mapping. While beta consistently became labeled under chase conditions (both in growing and mating cells), it was not clear whether it is part of the vivo processing event(s) which interrelates X, alpha, gamma, and delta. Beta was not recognized by alpha antibodies. Despite this uncertainty, these results suggest that proteolytic processing serves to generate significant changes in the complement of H1-like histones present in this nucleus.     

Electron-microscopical evidence that ribosomal chromatin of human circulating lymphocytes is devoid of histones

We have studied the distribution of histones in the nucleolus of human circulating lymphocytes in situ, using thin sections, either treated with antibodies against the core histones revealed by colloidal gold, or stained with the acrolein-silver methenamine technique for basic proteins. Gold particles were not found in the fibrillar centre, nor were silver-stained structures visible in this nucleolar component. Since the fibrillar centre contains the bulk of the ribosomal chromatin which is in a completely extended, non-nucleosomal configuration, our results indicate that this chromatin is devoid of histones.