Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an <Emphasis Type="Italic">ADA2</Emphasis>-independent Gcn5p activity

Background  

The acetylation of the core histone NH₂-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos

Lysine acetylation is an important post‐translational modification (PTM). Since the development of MS‐based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS‐based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine‐acetylated proteins in 5‐day postfertilization (hpf) embryos of D. rerio. Among lysine‐acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis.     

Structure and binding of the H4 histone tail and the effects of lysine 16 acetylation

The H4 histone tail plays a critical role in chromatin folding and regulation--it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The molecular mechanism associated with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of molecular dynamics simulations, molecular docking calculations, and free energy computations to investigate the structure of the H4 tail in solution, the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approximately -7 kcal mol(-1). We have identified the specific interactions that stabilize this binding as well as the associated energetics. The acetylation of K16 is found to reduce the α-helix forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-ranged electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via posttranslational modifications of histone tails, which could be of interest to chromatin biology, cancer, epigenetics, and drug design.     

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysyl‐tRNA synthetase‐tRNA^Pyl pair, azidonorleucine is genetically encoded in E. coli . Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site‐specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su‐H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post‐translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.     

A Genetically Encoded Allysine for the Synthesis of Proteins with Site‐Specific Lysine Dimethylation

Using the amber suppression approach, N^ϵ‐(4‐azidobenzoxycarbonyl)‐δ,ϵ‐dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site‐specific lysine dimethylation. Using this approach, dimethyl‐histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.     

Unexpected Crosslinking and Diglycation as Advanced Glycation End-Products from Glyoxal

Glyoxal-derived advanced glycation end-products (AGEs) are formed in physiological systems affecting protein/peptide function and structure. These AGEs are generated during aging and chronic diseases such as diabetes and are considered arginine glycating agents. Thus, the study of glyoxal-derived AGEs in lysine residues and amino acid competition is addressed here using acetylated and non-acetylated undecapeptides, with one arginine and one lysine residue available for glycation. Tandem mass spectrometry results from a Fourier transform ion cyclotron resonance mass spectrometer showed glycated species at both the arginine and lysine residues. One species with the mass addition of 116.01096 Da is formed at the arginine residue. A possible structure is proposed to explain this finding (Nδ-[2-(dihydroxymethyl)-2H,3aH,4H,6aH-[1, 3]dioxolo[5,6-d]imidazolin-5-yl]-L-ornithine-derived AGE). The second species corresponded to intramolecular crosslink involving the lysine residue and its presence is checked with ion-mobility mass spectrometry. Graphical Abstract

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Examining histone modification crosstalk using immobilized libraries established from ligation-ready nucleosomes

Chromatin signaling relies on a plethora of posttranslational modifications (PTM) of the histone proteins which package the long DNA molecules of our cells in reoccurring units of nucleosomes. Determining the biological function and molecular working mechanisms of different patterns of histone PTMs requires access to various chromatin substrates of defined modification status. Traditionally, these are achieved by individual reconstitution of single nucleosomes or arrays of nucleosomes in conjunction with modified histones produced by means of chemical biology. Here, we report an alternative strategy for establishing a library of differentially modified nucleosomes that bypasses the need for many individual syntheses, purification and assembly reactions by installing modified histone tails on ligation-ready, immobilized nucleosomes reconstituted in a single batch. Using the ligation-ready nucleosome strategy with sortase-mediated ligation for histone H3 and intein splicing for histone H2A, we generated libraries of up to 280 individually modified nucleosomes in 96-well plate format. Screening these libraries for the effects of patterns of PTMs onto the recruitment of a well-known chromatin factor, HP1 revealed a previously unknown long-range cross-talk between two modifications. H3S28 phosphorylation enhances recruitment of the HP1 protein to the H3K9 methylated H3-tail only in nucleosomal context. Detailed structural analysis by NMR measurements implies negative charges at position 28 to increase nucleosomal H3-tail dynamics and flexibility. Our work shows that ligation-ready nucleosomes enable unprecedented access to the ample space and complexity of histone modification patterns for the discovery and dissection of chromatin regulatory principles.

280 different patterns of histone modifications were installed in preassembled nucleosomes using PTS and SML enabling screening of readout crosstalk.     

Mapping Post-translational Modifications of Histones H2A, H2B and H4 in Schizosaccharomyces pombe

Core histones are known to carry a variety of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. The nature and biological functions of these PTMs in histones from plants, animals and budding yeast have been extensively investigated. In contrast, the corresponding studies for fission yeast were mainly focused on histone H3. In the present study, we applied LC-nano-ESI-MS/MS, coupled with multiple protease digestion, to identify PTMs in histones H2A, H2B and H4 from Schizosaccharomyces pombe (S. pombe), the typical model organism of fission yeast. Various protease digestions provided high sequence coverage for PTM mapping, and accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from tri-methylation. Many modification sites conserved in other organisms were identified in S. pombe. In addition, some unique modification sites, including N-terminal acetylation in H2A and H2B as well as K123 acetylation in H2A.β, were observed. Our results provide a comprehensive picture of the PTMs of histones H2A, H2B and H4 in S. pombe, which serves as a foundation for future investigations on the regulation and functions of histone modifications in this important model organism.     

Peptide mass mapping of acetylated isoforms of histone H4 from mouse lymphosarcoma cells treated with histone deacetylase (HDACs) inhibitors

The acetylated isoforms of histone H4 from mouse lymphosarcoma cells treated with HDAC inhibitors trichostatin A (TSA) and depsipeptide (DDP) were separated by acetic acid urea-polyacrylamide gel electrophoresis (AU-PAGE), in-gel digested, and analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The acetylation pattern of histone H4 in mouse lymphosarcoma cells induced by TSA was established in which acetylation initially occurred at K16 followed by K12 and then K8 and/or K5. An identical order of acetylation was found for cells treated with DDP.     

LSD1: more than demethylation of histone lysine residues

Lysine-specific histone demethylase 1 (LSD1) represents the first example of an identified nuclear protein with histone demethylase activity. In particular, it plays a special role in the epigenetic regulation of gene expression, as it removes methyl groups from mono- and dimethylated lysine 4 and/or lysine 9 on histone H3 (H3K4me1/2 and H3K9me1/2), behaving as a repressor or activator of gene expression, respectively. Moreover, it has been recently found to demethylate monomethylated and dimethylated lysine 20 in histone H4 and to contribute to the balance of several other methylated lysine residues in histone H3 (i.e., H3K27, H3K36, and H3K79). Furthermore, in recent years, a plethora of nonhistone proteins have been detected as targets of LSD1 activity, suggesting that this demethylase is a fundamental player in the regulation of multiple pathways triggered in several cellular processes, including cancer progression. In this review, we analyze the molecular mechanism by which LSD1 displays its dual effect on gene expression (related to the specific lysine target), placing final emphasis on the use of pharmacological inhibitors of its activity in future clinical studies to fight cancer.Subject terms: Epigenetics, Histone post-translational modifications