Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of Bombyx mori

Epigenetic information is encoded in post-translational modifications (PTMs) of histones. Various combinations of these marks contribute to the regulation of chromatin-templated DNA metabolisms. The histone code is gradually translated into biological responses in model organisms. However, in the silkworm, the modifications of histones with unique holocentric chromosomes have not yet been analyzed. TAU-PAGE analysis of the silkworm histone variants H2A, H2B, and H3, separated by RP-HPLC, suggested silkworm specific modification. Detailed mass spectrometry analyses of the peptides derived from the N-terminus of the silkworm H3.2 generated by glutamyl endopeptidase, lysyl endopeptidase, and trypsin digestions revealed global modifications around H3K9.     

Effects of posttranslational modifications on the structure and dynamics of histone H3 N-terminal Peptide

The highly conserved signature N-terminal peptide of histone protein H3 plays crucial roles in gene expression controls. We investigated the conformational changes of the peptide caused by lysine dimethylation and acetylation of the histone H3 N-terminal tail by molecular dynamics and replica-exchange molecular dynamics simulations. Our results suggest that the most populated structures of the modified H3 N-terminal peptides are very similar to those of the wild-type peptide. Thus, the modifications introduce marginal changes to the most favorable conformations of the peptides. However, the modifications have significant effects on the stabilities of the most populated states that depend on the modifications. Whereas dimethylation of lysine 4 or lysine 9 alone tends to stabilize the most populated states, double dimethylation and acetylation of both lysine 4 and lysine 9 reduce both the helical conformation and the stability of the most populated states significantly. The calculated melting temperatures showed that the doubly acetylated peptide has the lowest melting temperature (T_m = 324 K), which is notably lower than the melting temperatures of the other four peptides (T_m ≈ 346-350 K). In light of the existing experimental evidence, we propose that the changes in the dynamics of the modified variants contribute to their different functional roles.     

Sequence-dependent nucleosome structural and dynamic polymorphism. Potential involvement of histone H2B N-terminal tail proximal domain

Relaxation of nucleosomes on an homologous series (pBR) of ca 350-370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by approximately 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a approximately 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance ( approximately 20 bp) on H3-tail interactions with the small groove at the nucleosome entry-exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them.     

The effect of exposure to carcinogenic metals on histone tail modifications and gene expression in human subjects

The precise mechanisms by which nickel and arsenic compounds exert their carcinogenic properties are not completely understood. In recent years, alterations of epigenetic mechanisms have been implicated in the carcinogenesis of compounds of these two metals. In vitro exposure to certain nickel or arsenic compounds induces changes in both DNA methylation patterns, as well as, in the levels of posttranslational modifications of histone tails. Changes in DNA methylation patterns have been reported in human subjects exposed to arsenic. Here we review our recent reports on the alterations in global levels of posttranslational histone modifications in peripheral blood mononuclear cells (PBMCs) of subjects with occupational exposure to nickel and subjects exposed to arsenic in their drinking water. Occupational exposure to nickel was associated with an increase in H3K4me3 and decrease in H3K9me2. A global increase in H3K9me2 and decrease in H3K9ac was found in subjects exposed to arsenic. Additionally, exposure to arsenic resulted in opposite changes in a number of histone modifications in males when compared with females in the arsenic population. The results of these two studies suggest that exposure to nickel or arsenic compounds, and possibly other carcinogenic metal compounds, can induce changes in global levels of posttranslational histone modifications in peripheral blood mononuclear cells.     

The role of DNA methylation,nucleosome occupancy and histone modifications in paramutation

Paramutation is the transfer of epigenetic information between alleles that leads to a heritable change in expression of one of these alleles. Paramutation at the tissue‐specifically expressed maize (Zea mays) b1 locus involves the low‐expressing B′ and high‐expressing B‐I allele. Combined in the same nucleus, B′ heritably changes B‐I into B′. A hepta‐repeat located 100‐kb upstream of the b1 coding region is required for paramutation and for high b1 expression. The role of epigenetic modifications in paramutation is currently not well understood. In this study, we show that the B′ hepta‐repeat is DNA‐hypermethylated in all tissues analyzed. Importantly, combining B′ and B‐I in one nucleus results in de novo methylation of the B‐I repeats early in plant development. These findings indicate a role for hepta‐repeat DNA methylation in the establishment and maintenance of the silenced B′ state. In contrast, nucleosome occupancy, H3 acetylation, and H3K9 and H3K27 methylation are mainly involved in tissue‐specific regulation of the hepta‐repeat. Nucleosome depletion and H3 acetylation are tissue‐specifically regulated at the B‐I hepta‐repeat and associated with enhancement of b1 expression. H3K9 and H3K27 methylation are tissue‐specifically localized at the B′ hepta‐repeat and reinforce the silenced B′ chromatin state. The B′ coding region is H3K27 dimethylated in all tissues analyzed, indicating a role in the maintenance of the silenced B′ state. Taken together, these findings provide insight into the mechanisms underlying paramutation and tissue‐specific regulation of b1 at the level of chromatin structure.     

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ～10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.     

Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the "degree" of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.     

Selective radiolabelling and identification of a strong nucleosome binding site on the globular domain of histone H5.

We describe a chemical investigation of the nucleosome binding site(s) on histone H5. Selective radiolabelling by reductive methylation has led to the identification of lysine residues in H5 that are protected by its association with chromatin. The most strongly protected lysine is Lys-85 which occurs in the globular domain, in a region that is highly conserved between H5 and H1, and in H1 variants, and which probably constitutes a strong binding site for DNA where it enters and leaves the nucleosome. Lysines in the amino-terminal and lysine-rich carboxy-terminal tails are only weakly protected against chemical modification, suggesting a different mode of interaction with DNA.     

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.