Chromatin superstructure-dependent crosslinking with DNA of the histone H5 residues Thr1, His25 and His62

The points of histone H5 interactions with DNA within nucleosomes and chromatin at different levels of compaction are delineated by identification of H5 amino acid residues that can be covalently bound to DNA. Three major crosslinkable points of H5 are His25, His62 (both within the globular part of the molecule), and N-terminal Thr1. His25 interacts with the terminal regions of nucleosomal DNA; His62 appears to bind more distal segments of the linker DNA. The His25-DNA crosslink predominates in the isolated mononucleosomes and persists throughout the chromatin condensation states studied, from extended oligonucleosomal chains to nuclei. His62 is the strongest crosslinking site in nuclei; in oligonucleosomes, the predominance of the His62-DNA crosslink requires the number of nucleosomes in the chain to be above some critical value. The Thr1-DNA crosslink is generated only in decondensed poly- or oligonucleosomes, but not in mononucleosomes. Thus, underlying the higher-order folding transitions of the nucleosomal chain is the restructuring of H5-DNA interactions.     

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.     

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.     

Influence of Distamycin,Chromomycin, and UV-irradiation on Extraction of Histone H1 from Rat Liver Nuclei by Polyglutamic Acid

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A3 (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this dif- ference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and s~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (s~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV- stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (s~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.     

Histone modifications in Arabidopsis- high methylation of H3 lysine 9 is dispensable for constitutive heterochromatin

N-terminal modifications of nucleosomal core histones are involved in gene regulation, DNA repair and recombination as well as in chromatin modeling. The degree of individual histone modifications may vary between specific chromatin domains and throughout the cell cycle. We have studied the nuclear patterns of histone H3 and H4 acetylation and of H3 methylation in Arabidopsis. A replication-linked increase of acetylation only occurred at H4 lysine 16 (not for lysines 5 and 12) and at H3 lysine 18. The last was not observed in other plants. Strong methylation at H3 lysine 4 was restricted to euchromatin, while strong methylation at H3 lysine 9 occurred preferentially in heterochromatic chromocenters of Arabidopsis nuclei. Chromocenter appearance, DNA methylation and histone modification patterns were similar in nuclei of wild-type and kryptonite mutant (which lacks H3 lysine 9-specific histone methyltransferase), except that methylation at H3 lysine 9 in heterochromatic chromocenters was reduced to the same low level as in euchromatin. Thus, a high level of H3methylK9 is apparently not necessary to maintain chromocenter structure and does not prevent methylation of H3 lysine 4 within Arabidopsis chromocenters.     

Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.

Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1.     

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.     

The effects of salt concentration and H-1 depletion on the digestion of calf thymus chromatin by micrococcal nuclease.

We have removed histone H1 specifically from calf thymus nuclei by low pH treatment, and studied the digestion of such nuclei in comparison with undepleted nuclei. By a number of criteria the nuclei do not appear damaged. The DNA repeat-length in nuclear chromatin is found to be the same (192 +/- 4 bp) in the presence or absence of H1. These experiments demonstrate that the core histone complex of H2A, H2B, H3, and H4 can itself protect DNA sequences as long as 168 bp from nuclease. Our interpretation is that this represents an important structural element in chromatin, carrying two full turns of superhelical DNA. Depending on conditions of digestion this 168 bp fragment may be metastable and is normally rapidly converted by exonucleolytic trimming to the well-known "core-particle" containing 145 bp. Larger stable DNA fragments observed indigestion of H-1 depleted nuclei appear to arise from oligomers assembled from 168 bp cores in close contact exhibiting trimming of 0-20 bp at the ends. Electrophorograms of undepleted nuclear digests reveal oligomer bands in several size classes, each corresponding to one or more combinations of 168 bp particles, H1-protected spacers of about 20 bp length, and particles with ends trimmed to varying degrees.     

Protein kinase in HeLA nucleosomes: a reevaluation of the interactions of histomes with the ends of core particle DNA.

HeLa chromatin core particles contain a protein kinase which transfers phosphate from ATP to both nonhistone proteins and histones. The enzyme preferentially modifies H3 among the histones; about 7% of the H3 molecules in the nucleoprotein are modified at saturation. Activity of this kinase likely contributed to earlier results using crosslinking methodology to study which histones interact with the ends of core particle DNA. When the kinase is largely removed by sedimentation of core particles through sucrose gradients containing 0.45 M NaCl, crosslinking of the 5'-terminal label on DNA is observed only to histone H3. The overall efficiency of the crosslinking reaction is about 15%. The origin of the 5'-terminal 32P previously assigned as crosslinked to H4 is not explained by the current experiments.     

Higher order chromatin structure determines double-nucleosome periodicity of DNA fragmentation

Double-nucleosome periodicity of DNA fragmentation with DNAse I in the nuclei of cells differing in size of the linker DNA length and lysine-rich histone composition was analyzed by means of nondenaturing agarose gel electrophoresis. DNAse I revealed this type of periodicity in rat thymus and CHO cell nuclei as well as in erythrocyte nuclei. It has been deduced that the so-called nucleodisome structure is also typical of cells possessing a usual DNA repeat length (200 bp or less) and lysine-rich histone H1. Two probably related events are important for establishing a clear double-nucleosome periodicity of DNA fragmentation: the replacement of H1 histone by a specific arginine-rich histone fraction (H5 histone in the case of erythrocyte) and the increase of the linker DNA length. The results are interpreted in terms of supranucleosomal organization of chromatin which may determine the dinucleosome periodicity of DNA fragmentation due to a specific packing of nucleosomes.     

Inaccessibility of theEuplotes telomere binding protein

The telomere binding protein (TP) from the macronucleus of the ciliateEuplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequences. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augumented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

A lysine-rich protein functions as an H1 histone in Dictyostelium discoideum chromatin.

Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp). Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion. This was confirmed by stripping the protein from chromatin with Dowex resin. Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion. Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes. Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes. Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome. Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx. 10 kilodaltons. Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease. Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.     

Histone crosslinking patterns indicate dynamic binding of histone H1 in chromatin

Crosslinking of histone H1 molecules to each other and to the core histones with bifunctional reagents in mouse liver nuclei and chromatin was compared with that under the conditions of random 'contacts' between these molecules. The patterns of crosslinking of the H1 subfractions (H1A, H1B, and H10) to each other in nuclei, chromatin and in solution at different ionic strengths due to random collisions were essentially the same. Moreover, the contacts between the H1 molecules were qualitatively the same in nuclei, chromatin and in solution also at the level of the chymotryptic halves of the H1 molecules. The contacts between the H1 molecules and the core histones in nuclei were similar to those obtained in chromatin at 70 mM NaCl, when H1 molecules readily migrate, and at 0.6 M NaCl, when H1 molecules are dissociated from chromatin. We conclude that spatial arrangement of H1 subfractions and mutual orientation of H1 molecules in isolated nuclei are random-like at least in terms of cross-linking. The static and dynamic models of histone H1 binding to chromatin compatible with the known data are considered. Although unequivocal verification of the models is not possible at present, the dynamic models do correspond better to recent data on the location of the histone H1 in nuclei and chromatin.     

Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.     

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.)