Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.     

Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3.

Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones. This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4. Since H4 is hypoacetylated in heterochromatin we wished to determine whether acetylation is involved in regulating the contacts between SIR3 and H4. Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance. We find that acetylation of H4 lysines reduces binding (K(a)) of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide. Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin. These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes.     

Going global: Novel histone modifications in the globular domain of H3

Post-translational modifications (PTM) of histones are key regulators of chromatin function. New mass spectometrical technologies have revealed that PTMs are not restricted to the histone tails, but can also be found in the globular domains, especially at the DNA-binding surface of the nucleosomes. Recent work on this new group of epigenetic marks showed that these modifications have not only the potential to alter the physical properties of the nucleosome, but may act as signals that regulate the recruitment of effector proteins to chromatin as well.     

Crystallization of the globular domain of histone H5

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene that codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution. The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.     

Acetylation sites in histone H3 from Physarum polycephalum

Histone H3 from Physarum polycephalum was labelled with [³H]acetate in G2 phase of the cell cycle. Only histones H3 and H4 were labelled and the H4 was removed by chromatography. Sequential Edman degradation of labelled H3 showed that acetate was incorporated into residues 9, 14, 18 and 23 which correspond to the sites of acetyl-lysine determined in histones H3 from other organisms. The results confirm the sequence conservation of H3 and support the notion that data on H3 acetylation, obtained with Physarum, can be extrapolated to higher eukaryotes.     

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.     

The role of the central globular domain of histone H5 in chromatin structure

Histone H5 contains three tyrosines in the central, apolar region of the molecule. All three tyrosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain in essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result is a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higher-order chromatin structure.     

Co-operative binding of the globular domain of histone H5 to DNA.

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.     

Identification of two DNA-binding sites on the globular domain of histone H5.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA-binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X-ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in ''tramline'' complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild-type recombinant GH5. They still bind to H1/H5-depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA-binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.