The core histone N termini function independently of linker histones during chromatin condensation

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.     

Characterization of nucleosomes consisting of the human testis/sperm-specific histone H2B variant (hTSH2B)

We have reported earlier the occurrence of a specific histone H2B variant in human testis and sperm. Here we have structurally characterized this protein, its association with the rest of the histone octamer, and its effects on the nucleosome structure. We show that a reconstituted octamer consisting of hTSH2B and a stoichiometric complement of histones H2A, H3, and H4 exhibits a lower stability compared to the reconstituted native counterpart consisting of H2B. In contrast, the hTSH2B containing octamers are able to form nucleosome core particles which are structurally and dynamically indistinguishable from those reconstituted with octamers consisting of only native histones. Furthermore, the presence of hTSH2B in the nucleosome does not affect its ability to bind to linker histones.     

Role of histone tyrosines in nucleosome formation and histone-histone interaction

We have studied the functional properties of iodinated histones. Isolated, denatured histones were iodinated at trace levels and then renatured together with carrier histones and high molecular weight DNA to form nucleohistone. Nucleosomes were prepared from the reconstitute using micrococcal nuclease, and the relative representations of the individual iodinated tyrosines of the histones in the reconstituted nucleosomes were determined. Our principal findings are 1) that denatured histones can be iodinated at any tyrosine without interfering in subsequent nucleosome reconstitution and 2) that the resulting reconstituted nucleosomes nevertheless possess histone cores of altered stability, being either more or less stable depending on the particular tyrosine which is iodinated. We show that tyrosines 37, 40, and 42 of H2B are protected from iodination in intact core particles, as expected since these tyrosines lie within the H2B-H2A binding site. Yet iodination of these tyrosines in denatured H2B does not interfere with nucleosome assembly. However, the histone cores isolated from these reconstituted nucleosomes are of diminished stability as assayed by Sephadex column chromatography in 2 M salt. In contrast, iodination of tyrosines 83 and 121 of H2B, as well as iodination of the tyrosines of H2A, increases the stability of the histone octamer core. Iodination of H4 tyrosine 72 is without effect on histone octamer stability. Tyrosine iodination constitutes a profound amino acid alteration in the context of the absolute evolutionary conservation of most histone tyrosines. For example, all H2Bs sequenced to date, from fungi to mammals, possess tyrosines at positions 37, 40, and 42. Our results suggest that the immutability of these tyrosines reflects some sophisticated function of the nucleosome histone core beyond the assembly and mere maintenance of a compact structure.     

Reconstitution of hemisomes on budding yeast centromeric DNA

The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly.     

pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract

In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10. In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs. We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10. The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation. Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts. These data identify pEg2 as a good candidate for mitotic histone H3 kinase. However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs. This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae.     

Remodeling of sperm chromatin after fertilization involves nucleosomes formed by sperm histones H2A and H2B and two CS histone variants

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.     

Mitotic phosphorylation of histone H3 threonine 80

The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells.     

Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg²⁺, Ca²⁺, cobalt(III)hexammine³⁺, spermidine³⁺ and spermine⁴⁺] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K → Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.     

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)     

The NH2 tail of the novel histone variant H2BFWT exhibits properties distinct from conventional H2B with respect to the assembly of mitotic chromosomes

We have studied the functional and structural properties of nucleosomes reconstituted with H2BFWT, a recently identified putative histone variant of the H2B family with totally unknown function. We show that H2BFWT can replace the conventional histone H2B in the nucleosome. The presence of H2BFWT did not affect the overall structure of the nucleosome, and the H2BFWT nucleosomes exhibited the same stability as conventional nucleosomes. SWI/SNF was able to efficiently remodel and mobilize the H2BFWT nucleosomes. Importantly, H2BFWT, in contrast to conventional H2B, was unable to recruit chromosome condensation factors and to participate in the assembly of mitotic chromosomes. This was determined by the highly divergent (compared to conventional H2B) NH2 tail of H2BFWT. These data, in combination with the observations that H2BFWT was found by others in the sperm nuclei and appeared to be associated with the telomeric chromatin, suggest that H2BFWT could act as a specific epigenetic marker.     

Switching on Chromatin: Mechanistic Role of Histone H4-K16 Acetylation

DNA in eukaryotic organisms does not exist free in cells, but instead is present as chromatin, a complex assembly of DNA, histone proteins, and chromatin-associated proteins. Chromatin exhibits a complex hierarchy of structures, but in its simplest form it is composed of long linear arrays of nucleosomes. Nucleosomes contain 147 base pairs of DNA wrapped around a histone octamer, consisting of two copies each of histones H2A, H2B, H3 and H4, where 15-38 amino terminal residues of each histone protein extends past the DNA gyres to form histone “tails” 1. Chromatin provides a versatile regulatory platform for nearly all cellular processes that involve DNA, and improper chromatin regulation results in a wide range of diseases, including various cancers and congenital defects. One major way that chromatin regulates DNA utilization is through a wide range of post-translational modification of histones, including serine and threonine phosphorylation, lysine acetylation, methylation, ubiquitination, and sumoylation, and arginine methylation 2. Histone H4 K16 acetylation is a modification that occurs on the H4 histone tail and is one of the most frequent of the known histone modifications. We have demonstrated that this mark both disrupts formation of higher-order chromatin structure and changes the functional interaction of chromatin-associated proteins 3. Our results suggest a dual mechanism by which H4 K16 acetylation can ultimately facilitate genomic functions.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

H2A.Z stabilizes chromatin in a way that is dependent on core histone acetylation

The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3-H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.     

Phosphorylation of sea urchin histone CS H2A

Phosphorylation of cleavage stage (CS) histones was studied during the first cell cycle in male pronuclei of the sea urchin. Histone CS H2A rapidly incorporated 32PO4 during the replication period, but not before. Peptide mapping and amino acid analysis of radiolabelled CS H2A showed that phosphorylation occurred mainly on serine residues located in the C-terminal region of the molecule. When DNA replication was inhibited with aphidicolin both CS H2A and CS H2B accumulated in male pronuclei at the same rate as in the control culture, whereas accumulation of H3 and H4 histones was reduced. Incorporation of 32PO4 by CS H2A doubled when DNA synthesis was inhibited with aphidicolin. Thus phosphorylation of CS H2A was correlated with transport of CS histones from the egg storage pool to the male pronucleus, but not with chromatin synthesis, indicating that this event precedes nucleosome formation. A role for phosphorylation and dephosphorylation of the CS H2A C-terminal region in modulating transport of stored CS histone dimers and their assembly into nucleosomes is discussed.     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.     

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.     

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.     

Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers.

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.     

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.