Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.     

Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k _cat/K _m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K _m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.     

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides—viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein–Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4—were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10⁻⁶ M > K_D > 10⁻⁷ M) and interacted with HCP1 with higher apparent affinity (K_D = 10⁻⁸ M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.     

K8 and K12 are biotinylated in human histone H4.

Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, e.g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin-peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modifications. Substitution of a given lysine (positive charge) with glutamate (negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines.     

State of histone modification in the rat Ig-beta/growth hormone locus.

The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) at 11 targets in the rat Ig-beta/growth hormone locus. Marked enhancement of the acetylation of histones H3 and H4 and the dimethylation of H3 Lys4 was observed in the chromatin situated close to the promoter of an actively transcribed gene. Chromatin positioned near a cell-type-specific DNase I-hypersensitive site with enhancer activity had the same histone modifications as the active promoter. In one transcribed intron, chromatin with fewer histone modifications was found, and in another transcribed intron, chromatin with markedly enhanced modifications was found. In most cases, no appreciable difference in the acetylation of histones H3 and H4 was found at prominently enhanced targets. However, different acetylation levels of H3 and H4 were found at one target. The targets with enhanced dimethylation of the H3 Lys4 residue coincided with those with prominently enhanced acetylation of histones H3 and H4.     

Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos

Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. Our results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, our results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated.     

A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications

The N-terminal tails of the histones are subject to many enzyme-mediated post-translational modifications, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, poly-ADP ribosylation and the attachment of the small peptide ubiquitin. These modifications, singly or in combination, are thought to generate an epigenetic code that specifies different patterns of gene activity. We present a detailed study on the mapping of histone post-translational modifications using a combination of matrix-assisted laser desorption/ionization-time of flight and electrospray ionization tandem mass spectrometry analysis of peptides generated by protease cleavage of individual histones isolated from different developmental stages. Due to their high content in basic amino acid residues and in order to be able to quantitatively compare two different samples we developed a chemical derivatization protocol. This strategy enabled us to determine the primary sequence of the peptides and to unambiguously assign specific modifications. This method is generally applicable to histone samples from various sources and can be used to study changes of modification patterns during early embryonic development or tissue differentiation and regeneration.