Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles

Protein and DNA contributions in the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer) to a right-handed conformation have been investigated in a recent article from this laboratory. As the evidence for a protein contribution, a sterical hindrance introduced at the H3/H3 interface of the two constituent H3-H4 dimers by oxidation of H3 cysteine 110 blocked the tetramer in a half-left-handed or semi-right-handed conformation, depending on the SH-reagent used. The DNA contributed at the level of the dyad region, which appeared to act through its sequence-dependent deformability in modulating both the loop threshold positive constraint required to trigger the transition, and the tetrasome lateral opening. This opening, which electron microscopic visualizations directly showed to be associated with the transition, is expected to help remove the clash between the entering and exiting DNAs. In this work, the transition mechanism was further investigated by applying a positive constraint in the loop through ethidium bromide (EtBr) intercalation. This technique, including the determination of binding isotherms, has first been used with mononucleosomes on DNA minicircles, and has revealed that these particles could tolerate large positive supercoilings without disruption, owing to the loop ability to cross positively in a histone tail-dependent manner. The transition of 359 bp tetrasomes was found to go to completion in lower salt (10 mM), but not in higher salt (100 mM), whereas the transition of 256 bp tetrasomes was already hindered in lower salt. Histone acetylation relieved that lower salt hindrance but enhanced the higher salt hindrances. These data again pointed to the DNA in the dyad region as a regulator of the transition. The block was indeed expected to originate from a local EtBr intercalation in that DNA, which opposed its overtwisting during the transition. The occurrence of the block, or its relief, then depended on the outcome of the competition between the tails and EtBr for binding to that region, that is, on whether the tails could prevent EtBr intercalation before the ongoing transition hampered both bindings. Destabilization of the tails in the course of the transition is documented in an accompanying article through a relaxation study of a 351-366 bp tetrasome series.     

Nucleosome dynamics. VI. Histone tail regulation of tetrasome chiral transition. A relaxation study of tetrasomes on DNA minicircles

We have recently described the relaxation of mononucleosomes on an homologous series of 351-366 bp DNA minicircles, as a tool to study nucleosome structure and dynamics in vitro. Nucleosomes were found to have a tail-regulated access to three distinct DNA conformations, depending on the crossing between the entering and exiting DNAs, and its polarity. This approach was now used to explore tetrasome chiral transition, and the influence of the histone tails. The data confirmed the existence of two states, with linking number differences DeltaLk(t)=-0.74(+/-0.01) and +0.51(+/-0.06). As expected, the particle free energy is higher in the right-handed state (DeltaG(t)=1.9(+/-0.I) kT), but it decreased (to 1.3(+/-0.1) kT) upon histone acetylation and the addition of phosphate, a potent tail destabilizer. Removal of the tails with trypsin further decreased DeltaG(t) (to 0.6 kT), and also induced a loss of supercoiling in both states, to DeltaLk(t)=-0.64(+/-0.03) and +0. 35(+/-0.05). The loop end-conditions, and hence the parameters of the DNA superhelix, were then calculated for both states using the explicit solutions to the equations of the mechanical equilibrium in the theory of elastic rod model for DNA. Whereas the pitch of the DNA superhelix may be approximately equal and opposite in the two conformations, its radius (r) was 20% larger in the right-handed conformation, confirming previous observations by electron microscopy of a tetrasome lateral opening in that conformation. The above supercoiling losses were found to reflect a further 3 % increase in r (to 23 %) upon removal of the tails in the right-handed conformation, and a 14 % increase in the left-handed conformation. The use of composite tetramers with one histone tail intact and the other removed showed these effects to be essentially due to the H3 tails. Altogether, these results show that the H3 tails oppose the tetrasome opening which is expected to be required to relieve the clash between the entering and exiting DNAs in the course of the transition, but which also appears to be intrinsic to the protein reorientation mechanism. We propose that the block against opening results from the H3 tails intercalating into the small groove of the double helix at +/-10 bp from the dyad, and acting as wedges against local DNA straightening. The tails (especially H3) may therefore regulate tetrasome chiral transition in vivo.     

Spatial organization of the (H3-H4-H2A-H2B)2 histone octamer

Structure of the (H2A-H2B-H3-H4)2 histone octamer isolated from calf thymus chromatin at ionic strength 0.1 to 4.0 M NaCl, pH 7.6, was studied spectrofluorometrically. Sensitivity of lambda max tyrosine fluorescence position to structural changes of histone oligomers and to the processes of their association was shown. It were detect two ranges of cooperative changes in histone optical parameters at 0.6-1.4 M NaCl (transition I) and at 2.4-3.4 M NaCl (transition II): Transition I corresponds to the formation of equilibrium system (hexamer) + (dimer) in equilibrium octamer. Transition II corresponds to the structural changes of the histone octamer. Thus, fluorescence anisotropy increases, lambda max for fluorescence spectrum is shifted to the longer wavelengths, contributions of two components to fluorescence decay change, a fraction of fluorescence accessible to the quenching by I- decreases. Histone octamer formation is characterized by making specific contacts between the (H2A-H2B) dimer and (H3-H4)2 tetramer. These contacts are realized at gradual changing of ionic strengths (by dialysis). In the case of abrupt local changes of the environment the process is irreversibly shifted to formation of unspecific high molecular aggregates. The important function role for energetically degenerated states of histone oligomers, energy barriers between which can be overcome by changing total conditions of histone microenvironment in chromatin is discussed.     

Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)₂ tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)₂ tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)₂ tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.     

Association of nucleosome core particle DNA with different histone oligomers. Transfer of histones between DNA-(H2A,H2B) and DNA-(H3,H4) complexes

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.     

A rapid method of preparing the (H3-H4-H2A-H2b)(2) histone octamer in large quantities]

A simple and fast method for isolation of large amounts of the histone octamer (H2A-H2B-H3-H4)2 is proposed. This method is based on chromatin adsorption by hydroxyapatite with subsequent extraction of the histone octamer with 50 mM sodium-phosphate buffer containing 4 M NaCl pH 8.0. It was shown that the properties of the histone octamer isolated by this extractive procedure are identical with those of the histone octamer obtained by elution on a Sephadex G-100 column. The histone tetramer (H3-H4)2 and dimer (H2A-H2B) were obtained after gel filtration on Sephadex G-100 in 50 mM sodium-acetate (pH 5.6).     

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.     

Associative behavior of the histone (H3-H4)2 tetramer: dependence on ionic environment

Mixtures of histones H3 and H4 were examined by analytical ultracentrifugation and circular dichroism to determine their association behavior and secondary structure content in high and low ionic strength solvents containing chloride, phosphate, or sulfate. H3 and H4 were also cross-linked by using DSP in order to directly trap any intermolecular interactions occurring in solution. While H3 and H4 can exist as an H3-H4 dimer under limited conditions, they behave as a stable (H3-H4)2 tetramer under most conditions, particularly those which are physiologically relevant. In chloride-containing solutions, the equilibrium between H3-H4 and (H3-H4)2 is responsive to changes in ionic strength and paralleled by large changes in alpha-helicity. In sulfate- and phosphate-containing solutions, the equilibrium is again governed by ionic strength, but there are no significant changes in secondary structure accompanying shifts in the equilibrium. Small oligomers can be formed in the presence of sulfate and phosphate and trapped by the cross-linking reagent; these oligomers are much smaller than those formed in chloride-containing solutions. However, addition of the H2A-H2B dimer into the system prevents aggregation of the (H3-H4)2 tetramer by acting as a "molecular cap" and thus regulating the assembly pathway toward the formation of tripartite octamers. The observed assembly of H3 and H4 into a stable, tetrameric complex supports the concept of the core histone octamer having a tripartite organization in solution rather than being organized as two heterotypic tetramers.     

On the native structure of the histone H3-H4 complex.

Electrophoretic and sedimentation velocity studies on the histone H3–H4 complex show that provided the H3 cysteine residues remain reduced the complex reforms quantitatively when removed from a variety of denaturing conditions. If histone H3 is allowed to become intramolecularly oxidized while denatured only monomer and large aggregates are formed on return to native conditions. At pH 7 ionic strength 0.1 the complex remains with reduced sulfhydryl groups indefinitely suggesting a vital role for the sequence 96–110 in histone H3 in the tertiary structure of the complex.     

DNA methylation, histone H3 methylation, and histone H4 acetylation in the genome of a crustacean.

In this work, we used antibodies against histone H3 trimethylated at lysine 9 (H3K9m3); against histone H4 acetylated at lysines 5, 8, 12, and 16 (H4ac); and against DNA methylated at 5C cytosine (m5C) to study the presence and distribution of these markers in the genome of the isopod crustacean Asellus aquaticus. The use of these 3 antibodies to immunolabel spermatogonial metaphases yields reproducible patterns on the chromosomes of this crustacean. The X and Y chromosomes present an identical banding pattern with each of the antibodies. The heterochromatic telomeric regions and the centromeric regions are rich in H3K9m3, but depleted in m5C and H4ac. Thus, m5C does not seem to be required to stabilize the silence of these regions in this organism.     

Roles of the histone H2A-H2B dimers and the (H3-H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex

SWI-SNF is an ATP-dependent chromatin remodeling complex required for expression of a number of yeast genes. Previous studies have suggested that SWI-SNF action may remove or rearrange the histone H2A-H2B dimers or induce a novel alteration in the histone octamer. Here, we have directly tested these and other models by quantifying the remodeling activity of SWI-SNF on arrays of (H3-H4)(2) tetramers, on nucleosomal arrays reconstituted with disulfide-linked histone H3, and on arrays reconstituted with histone H3 derivatives site-specifically modified at residue 110 with the fluorescent probe acetylethylenediamine-(1,5)-naphthol sulfonate. We find that SWI-SNF can remodel (H3-H4)(2) tetramers, although tetramers are poor substrates for SWI-SNF remodeling compared with nucleosomal arrays. SWI-SNF can also remodel nucleosomal arrays that harbor disulfide-linked (H3-H4)(2) tetramers, indicating that SWI-SNF action does not involve an obligatory disruption of the tetramer. Finally, we find that although the fluorescence emission intensity of acetylethylenediamine-(1,5)-naphthol sulfonate-modified histone H3 is sensitive to octamer structure, SWI-SNF action does not alter fluorescence emission intensity. These data suggest that perturbation of the histone octamer is not a requirement or a consequence of ATP-dependent nucleosome remodeling by SWI-SNF.     

Complexes of the arginine-rich histone tetramer (H3)2(H4)2 with negatively supercoiled DNA: electron microscopy and chemical cross-linking.

Tetramers of the arginine-rich histones H3 and H4 associate with supercoiled SV40 DNA either singly, giving tetrameric nucleoprotein complexes or in pairs giving octameric complexes, both of which are visualized as beads in the electron microscope. The relative amounts of the two complexes may be revealed by complete cross-linking of the proteins, followed by analysis in SDS-polyacrylamide gels. By electron microscopy of unmodified and of cross-linked complexes, both the tetrameric and the octameric complexes are shown to have a diameter of 8-9 nm and to contain about 145 base pairs (a nucleosome core length) of DNA. The compaction of the DNA in both cases is thus similar to that in the nucleosome, which has a diameter of about 12.5 nm and contains 200 base pairs of DNA.     

Nucleosome cores containing H2B,H3, H4 and HMG2 are reconstituted

Nucleosome cores mixed with the high mobility group proteins, HMG1 and HMG2, in 2 M NaCl, 5 M urea, 0.2 mM EDTA and 10 mM Tris pH 7.0, have been reconstituted by salt gradient dialysis. The reconstituted material, in 10 mM Tris pH 7.0, had a sedimentation peak at the same position as that of control nucleosome cores in sucrose density gradient ultracentrifugation. The SDS polyacrylamide gel electrophoresis of the reconstituted nucleosome cores demonstrated that they contain H2B, H3, H4 and HMG2 and are selectively deficient in H2A. The circular dichroism of DNA of the reconstituted cores was indistinguishable from that of control nucleosome cores. The results suggest that HMG2 replaces H2A as a component of the nucleosome histone core during reconstitution.     

Histone-histone interactions. II. Structural stability of the histone H3-H4 complex.

The stability of the histone H3-H4 complex toward urea, changes in pH and ionic strength, and certain chemical modifications have been examined by gel electrophoresis anc circular dichronism. When uncomplexed, the two cysteine residues of histone H3 become rapidly oxidized, forming an intramolecular disulfide bridge which apparently blocks complex formation on return to complexing conditions. The complex was found to be unstable toward low values of pH and ionic strength, concentrations of urea exceeding 1 M, modifications of the cysteine residues, and fragmention in which the C terminal portions of either H3 or H4 are removed. A possible structure for this complex is proposed.     

Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain

DNA methylation and histone H4 acetylation play a role in gene regulation by modulating the structure of the chromatin. Recently, these two epigenetic modifications have dynamically and physically been linked. Evidence suggests that both modifications are involved in regulating imprinted genes - a subset of genes whose expression depends on their parental origin. Using immunoprecipitation assays, we investigate the relationship between DNA methylation, histone H4 acetylation and gene expression in the well-characterised imprinted Igf2-H19 domain on mouse chromosome 7. A systematic regional analysis of the acetylation status of the domain shows that parental-specific differences in acetylation of the core histone H4 are present in the promoter regions of both Igf2 and H19 genes, with the expressed alleles being more acetylated than the silent alleles. A correlation between DNA methylation, histone hypoacetylation and gene repression is evident only at the promoter region of the H19 gene. Treatment with trichostatin A, a specific inhibitor of histone deacetylase, reduces the expression of the active maternal H19 allele and this can be correlated with regional changes in acetylation within the upstream regulatory domain. The data suggest that histone H4 acetylation and DNA methylation have distinct functions on the maternal and paternal Igf2-H19 domains.