Centromere silencing and function in fission yeast is governed by the amino terminus of histone H3

BACKGROUND: Centromeric domains often consist of repetitive elements that are assembled in specialized chromatin, characterized by hypoacetylation of histones H3 and H4 and methylation of lysine 9 of histone H3 (K9-MeH3). Perturbation of this underacetylated state by transient treatment with histone deacetylase inhibitors leads to defective centromere function, correlating with delocalization of the heterochromatin protein Swi6/HP1. Likewise, deletion of the K9-MeH3 methyltransferase Clr4/Suvar39 causes defective chromosome segregation. Here, we create fission yeast strains retaining one histone H3 and H4 gene; the creation of these strains allows mutation of specific N-terminal tail residues and their role in centromeric silencing and chromosome stability to be investigated. RESULTS: Reduction of H3/H4 gene dosage to one-third does not affect cell viability or heterochromatin formation. Mutation of lysines 9 or 14 or serine 10 within the amino terminus of histone H3 impairs centromere function, leading to defective chromosome segregation and Swi6 delocalization. Surprisingly, silent centromeric chromatin does not require the conserved lysine 8 and 16 residues of histone H4. CONCLUSIONS: To date, mutation of conserved N-terminal residues in endogenous histone genes has only been performed in budding yeast, which lacks the Clr4/Suvar39 histone methyltransferase and Swi6/HP1. We demonstrate the importance of conserved residues within the histone H3 N terminus for the maintenance of centromeric heterochromatin in fission yeast. In sharp contrast, mutation of two conserved lysines within the histone H4 tail has no impact on the integrity of centromeric heterochromatin. Our data highlight the striking divergence between the histone tail requirements for the fission yeast and budding yeast silencing pathways.     

The bromodomain of Gcn5p interacts in vitro with specific residues in the N terminus of histone H4

Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear. Using a glutathione S-transferase pull down assay we show that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B. The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini. The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis. The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain. These residues differ from those known to be acetylated or to be involved in binding the SIR proteins. This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain.     

Chromatin-modifiying enzymes are essential when the Saccharomyces cerevisiae morphogenesis checkpoint is constitutively activated

Hsl7p plays a central role in the morphogenesis checkpoint triggered when yeast bud formation is impaired and is proposed to function as an arginine methyltransferase. HSL7 is also essential in the absence of the N-terminal tails of histones H3 or H4. The requirement for H3 and H4 tails may indicate a need for their post-translational modification to bypass the morphogenesis checkpoint. In support of this, the absence of the acetyltransferases Gcn5p or Esa1p, the deacetylase Rpd3p, or the lysine-methyltransferase Set1p resulted in death or extreme sickness in hslDelta mutants. These synthetic interactions involved both the activity of the chromatin-modifying enzymes and the complexes through which they act. Newly reported silencing phenotypes of hsl7Delta mirror those previously reported for gcn5Delta and rpd3Delta, thereby strengthening their functional links. In addition, synthetic interactions and silencing phenotypes were suppressed by inactivation of the morphogenesis checkpoint, either by SWE1 deletion or by preventing Cdc28p phosphorylation. A catalytically dead Hsl7p retained wild-type interactions, implying that modification of histone H3 or H4 N termini by Gcn5p, Esa1p, Rpd3p, and Set1p, but not by Hsl7p, was needed to bypass the morphogenesis checkpoint.     

Structural cooperativity in histone H3 tail modifications

Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

The many faces of histone lysine methylation

Diverse post-translational modifications of histone amino termini represent an important epigenetic mechanism for the organisation of chromatin structure and the regulation of gene activity. Within the past two years, great progress has been made in understanding the functional implications of histone methylation; in particular through the characterisation of histone methyltransferases that direct the site-specific methylation of, for example, lysine 9 and lysine 4 positions in the histone H3 amino terminus. All known histone methyltransferases of this type contain the evolutionarily conserved SET domain and appear to be able to stimulate either gene repression or gene activation. Methylation of H3 Lys9 and Lys4 has been visualised in native chromatin, indicating opposite roles in structuring repressive or accessible chromatin domains. For example, at the mating-type loci in Schizosaccharomyces pombe, at pericentric heterochromatin and at the inactive X chromosome in mammals, striking differences between these distinct marks have been observed. H3 Lys9 methylation is also important to direct additional epigenetic signals such as DNA methylation--for example, in Neurospora crassa and in Arabidopsis thaliana. Together, the available data strongly establish histone lysine methylation as a central modification for the epigenetic organisation of eukaryotic genomes.     

Yng2p-dependent NuA4 histone H4 acetylation activity is required for mitotic and meiotic progression.

In all eukaryotes, multisubunit histone acetyltransferase (HAT) complexes acetylate the highly conserved lysine residues in the amino-terminal tails of core histones to regulate chromatin structure and gene expression. One such complex in yeast, NuA4, specifically acetylates nucleosome-associated histone H4. Recent studies have revealed that NuA4 comprises at least 11 subunits, including Yng2p, a yeast homolog of the candidate human tumor suppressor gene, ING1. Consistent with prior data, we find that cells lacking Yng2p are deficient for NuA4 activity and are temperature-sensitive. Furthermore, we show that the NuA4 complex is present in the absence of Yng2p, suggesting that Yng2p functions to maintain or activate NuA4 HAT activity. Sporulation of diploid yng2 mutant cells reveals a defect in meiotic progression, whereas synchronized yng2 mutant cells display a mitotic delay. Surprisingly, genome-wide expression analysis revealed little change from wild type. Nocodazole arrest and release relieves the mitotic defects, suggesting that Yng2p may have a critical function prior to or during metaphase. Rather than a uniform decrease in acetylated forms of histone H4, we find striking cell-to-cell heterogeneity in the loss of acetylated histone H4 in yng2 mutant cells. Treating yng2 mutants with the histone deacetylase inhibitor trichostatin A suppressed the mitotic delay and restored global histone H4 acetylation, arguing that reduced H4 acetylation may underlie the cell cycle delay.