Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

CENP-A-containing nucleosomes: easier disassembly versus exclusive centromeric localization

CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-containing nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the alpha-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(+/-2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)2 tetramer was also more readily destabilized with heparin than the (H3-H4)2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, especially as is seen following CENP-A transient over-expression.     

Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.     

Atomic force microscopy sees nucleosome positioning and histone H1-induced compaction in reconstituted chromatin.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.     

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.     

The interaction of cisplatin and analogues with DNA in reconstituted chromatin

The influence of chromatin structure on cis-diamminedichloroplatinum(II) (cisplatin) DNA damage was investigated in a reconstituted nucleosome system. Nucleosomes were reconstituted on the somatic 5S rRNA gene from Xenopus borealis using the octamer transfer method of reconstitution. Footprinting techniques, utilising bleomycin and DNase I as the damaging agents, were employed to establish the precise location of positioned nucleosomes with respect to the DNA sequence. Reconstituted nucleosomal DNA was treated with cisplatin and drug-induced DNA adduct formation was quantitatively analysed with a polymerase stop assay using Taq DNA polymerase. A densitometric comparison of the relative damage band intensities between purified and reconstituted DNA revealed regions of relative protection corresponding to the sites of the positioned nucleosome cores. This indicated that the preferred site of cisplatin DNA binding was in the linker region of the nucleosome. Statistical analysis showed significant protection from cisplatin DNA damage in the core region of the nucleosome. Three cisplatin analogues were also investigated in this reconstituted nucleosome system. These analogues, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-dichlorobis(cyclohexylamine)platinum(II) (cis-[PtCl(2)(C(6)H(11)NH(2))(2)]) and dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-4-carboxamide)platinum(II) (ac-PtenCl(2)(n3)), were also found to target the linker region of the nucleosome. The latter DNA-targeted acridine-platinum complex gave rise to the most predominant footprints of all the Pt compounds tested.     

HMG-D and histone H1 alter the local accessibility of nucleosomal DNA

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

Sir2 deacetylates histone H3 lysine 56 to regulate telomeric heterochromatin structure in yeast

At telomeric heterochromatin in yeast, the Sir protein complex spreads from Rap1 sites to silence adjacent genes. This cascade is believed to occur when Sir2, an NAD(+)-dependent enzyme, deacetylates histone H3 and H4 N termini, in particular histone H4 K16, enabling more Sir protein binding. Lysine 56 of histone H3 is located at the entry-exit points of the DNA superhelix surrounding the nucleosome, where it may control DNA compaction. We have found that K56 substitutions disrupt silencing severely without decreasing Sir protein binding at the telomere. Our in vitro and in vivo data indicate that Sir2 deacetylates K56 directly in telomeric heterochromatin to compact chromatin and prevent access to RNA polymerase and ectopic bacterial dam methylase. Since the spread of Sir proteins is necessary but not sufficient for silencing, we propose that silencing occurs when Sir2 deacetylates H3 K56 to close the nucleosomal entry-exit gates, enabling compaction of heterochromatin.     

Supercoiling energy and nucleosome formation: the role of the arginine-rich histone kernel.

We have formed complexes of relaxed closed circular Col E1 DNA with various combinations of histones, and examined the effects of treating the complexes with nicking-closing enzyme. Germond et al (1) have shown that when a mixture of the four core histones of the nucleosome (HIA, H2B, H3 and H4) is used in such an experiment, the subsequently isolated DNA is supercoiled. We find that the arginine-rich histone pair, H3 and H4, is sufficient to induce the supercoiling observed in this experiment. Both H3 and H4 are required, and in the absence of either, no other histones are effective. H3 and and H4 are as efficient, per unit weight, as a mixture of the four histones in inducing supercoils. We also show that there is a large difference between the DNA bending energy needed to form a nucleosome and that needed to form one turn of normal superhelical DNA. These two processes are energetically quite distinct and probably separable. We estimate the free energy of interaction between DNA-bound histone pairs, and find that one or two such interactions would generate enough energy to fold the DNA into a nucleosome.