Histone N-terminal Tails Research Articles

A Novel Motif in Fungal Class 1 Histone Deacetylases Is Essential for Growth and Development ofAspergillus

Acetylation of the N-terminal tails of core histones is an important regulatory mechanism in eukaryotic organisms. In filamentous fungi, little is known about the enzymes that modify histone tails. However, it is increasingly evident that histone deacetylases and histone acetyltransferases are critical factors for the regulation of genes involved in fungal pathogenicity, stress response, and production of secondary metabolites such as antibiotics or fungal toxins. Here, we show that depletion of RpdA, an RPD3-type histone deacetylase of Aspergillus nidulans, leads to a pronounced reduction of growth and sporulation of the fungus. We demonstrate that a so far unnoticed motif in the C terminus of fungal RpdA histone deacetylases is required for the catalytic activity of the enzyme and consequently is essential for the viability of A. nidulans. Moreover, we provide evidence that this motif is also crucial for the survival of other, if not all, filamentous fungi, including pathogens such as Aspergillus fumigatus or Cochliobolus carbonum. Thus, the extended C terminus of RpdA-type enzymes represents a promising target for fungal-specific histone deacetylase-inhibitors that may have potential as novel antifungal compounds with medical and agricultural applications.

Phosphorylation of Histone H3 by Protein Kinase C Signaling Plays a Critical Role in the Regulation of the Developmentally Important TBX2 Gene

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

Over-expression of maize transglutaminase in tobacco chloroplasts

Histone N-terminal tails are subject to a variety of covalent modifications that ultimately affect chromatin structure. One such modification is N-methylation, which occurs on Lys (K) and Arg (R) amino acids. The enzymes performing these modifications are the histone N-methyl transferases (HMTs) that catalyze the transfer of a methyl group from s-adenosylmethionine (SAM) to the ɛ-amino groups of lysine and/or arginine residues within histones. HMTs can be divided into two groups—namely, (1) histone lysine N-methyltransferases and (2) histone arginine N-methyltransferases. All of the enzymes known to methylate lysines within histone N-terminal tails contain a conserved methyltransferase domain termed an SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain. In vivo methylated lysines may be found in the mono-, di-, or trimethylated State. It is becoming clear that these states of methylation have differing effects with respect to chromatin structure and transcription. Perhaps the most powerful tools in the study of histone methylation are site-specific antibodies capable of discriminating between the different methylated forms. These can then be used in numerous analyses aimed at unraveling the complexities of histone methylation. Of course, analysis of the HMTs themselves is often desirable. The chapter also describes various methods and protocols that are useful in the study of the enzymatic activity associated with histone methyltransferases.

Counterion Induced Electrostatic Condensation Of Nucleosomes And Chromatin Arrays

Recombinant molecular biology techniques have enabled preparation of pure and well-defined nucleosome core particles (NCP) and chromatin arrays. The 12_177_601 DNA sequence yields chromatin arrays with 12 histone octamers that can be studied with biophysical methods. The importance of the basic N-terminal histone tails can be investigated. The multivalent salt induced compaction of chromatin arrays and the aggregation of arrays, mononucleosomes (177 bp) and NCPs (147 bp), have been studied and compared with computer modelling using coarse-grained models.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe results show that the critical concentration of multivalent counterion concentration needed for compaction or aggregation, for all three systems follow the order Na+ ≈ K+ >> Mg2+ ≈ Ca2+ >> spermidine3+ > Co(NH3)63+ > spermine4+. This is in accordance with polyelectrolyte theory and a mechanism of condensation due to salt screening, attractive ion correlation and histone tail bridging, which in theoretical modelling must be described incorporating explicit mobile ions (beyond Debye-Huckel). The figure shows analytical ultracentrifugation sedimentation velocity measurements during titration of the chromatin array with Co(NH3)63+ inducing compaction. The inset demonstrates the compaction in coarse-grained computer modelling with explicit Co(NH3)63+ ions (right).

Inhibition of histone deacetylase causes reduction of appressorium formation in the rice blast fungus Magnaporthe oryzae

Post-translational modifications (PTMs) are important for cellular functions. The regulation of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is one of important PTMs for epigenetic control, protein activity and protein stability. The regulation of acetylation of the N-terminal histone tails of core histone affects gene expression. Two class I HDAC genes and two class II HDAC genes have been identified in the Magnaporthe oryzae genome. Treatment with Rpd3/Hda1 family (classical) HDAC inhibitor inhibited the appressorium differentiation of M. oryzae. Treatment with trichostatin A, a classical HDAC inhibitor, also decreased pathogenesis. Furthermore, analyses of HDAC mutants indicated that MoHda1 and MoHos2 were required for vegetative growth and conidiation, and MoHos2 was required for appressorium formation. Disruption MoRPD3 was unsuccessful, as in the case with Aspergillus nidulans RpdA. These data indicated that HDACs have important roles in the asexual differentiation of M. oryzae.

Histone N-terminal Tails Interfere with Nucleosome Traversal by RNA Polymerase II

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.

Loss of ATAC-specific acetylation of histone H4 at Lys12 reduces binding of JIL-1 to chromatin and phosphorylation of histone H3 at Ser10

Various combinations of post-translational modifications of the N-terminal tails of nucleosomal histones serve as signals to govern chromatin-related processes. The relationship, however, among different types of histone modifications - most frequently acetylation, phosphorylation and methylation - and the order of their establishment has been explored only in a few cases. Here we show that a reduced level of histone H4 acetylated at Lys12 by the ATAC-HAT complex leads to a decrease in the histone H3 phosphorylation at Ser10 by the kinase JIL-1. As JIL-1 activity antagonizes histone H3 dimethylation at Lys9 by SU(VAR)3-9, our observations demonstrate the interdependent actions of an acetyltransferase, a kinase and a methyltransferase. We demonstrate that, in accord with the steps of modifications, mutations that affect ATAC subunits (such as dGcn5, dAda2a and dAda3) (1) decrease the level histone H3 phosphorylation at Ser10, (2) can be rescued partially by JIL-1 overproduction, (3) enhance the spread of histone H3 dimethylation at Lys9 and (4) are suppressed by mutations of Su(var)3-9. We propose that a reduced level of histone H4 acetylated at Lys12 by ATAC attenuates histone H3 phosphorylation at Ser10 by JIL-1 owing to reduced binding of JIL-1 to hypoacetylated chromatin.

Formaldehyde-induced histone modifications in vitro.

Numerous experiments have demonstrated the genotoxic and mutagenic effects of formaldehyde, including DNA-protein cross-links (DPC). Histone was reported to be involved in the formation of DPC in which the epsilon-amino groups of lysine and exocyclic amino groups of DNA were thought to be cross-linked through multiple step reactions. Using mass spectrometry, the N-terminus of histone and lysine residues located in both the histone N-terminal tail and the globular fold domain were identified as binding sites for formaldehyde in the current study. The observation that only lysine residues without post-translational modification (PTM) can be attacked by formaldehyde indicates that PTM blocks the reaction between lysine and formaldehyde. Additionally, we found that formaldehyde-induced Schiff bases on lysine residues could inhibit the formation of PTM on histone, raising the possibility that formaldehyde might alter epigenetic regulation.

The role of histone H2A and H2B post-translational modifications in transcription: A genomic perspective

In eukaryotic cells, the genome is packaged with histones H2A, H2B, H3, and H4 to form nucleosomes. Each of the histone proteins is extensively post-translationally modified, particularly in the flexible N-terminal histone tail domains. Curiously, while post-translational modifications in histone H3 and H4 have been extensively studied, relatively little is known about post-translational modifications in the N-terminal domains of histone H2A and H2B. In this review, we will summarize current knowledge of post-translational modifications in the N-terminal domains of histone H2A and H2B, and the histone variant H2AZ. We will examine the distribution of these modifications in genomic chromatin, and the function of these modifications in transcription.

The Activity of HDAC8 Depends on Local and Distal Sequences of Its Peptide Substrates

This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8−19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16−19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression.

Involvement of fission yeast Clr6-HDAC in regulation of the checkpoint kinase Cds1

Modification of the N-terminal tail of histones is required for various nuclear processes. Here, we show that fission yeast Clr6-HDAC (histone deacetylase) regulates the checkpoint kinase Cds1 when DNA replication encounters a stressful condition. We found that the global level of acetylation of histone H4 was constant throughout the normal cell cycle, but was reduced significantly when the cell recovered from the HU-induced cell cycle arrest (or slow DNA replication). We identified the Clr6-HDAC as a component responsible for the reduction in the level of the H4 acetylation. Although DNA replication was completed, the HU-induced cell cycle arrest could not be released even after removal of HU in the clr6-1 mutant. Under this experimental condition, Cds1 kinase was maintained active and remained bound tightly to chromatin. We also demonstrated that Cds1 was active even after treatment with caffeine, an inhibitor for ATM/ATR that is an activator of Cds1. These results indicate that inactivation of Cds1 requires functional Clr6-HDAC independently of the conventional DNA replication checkpoint. When DNA replication is impeded, Clr6-HDAC activity may monitor damage on chromatin structure/environment, which is required for inactivation of Cds1.

Histone Modifications and Nuclear Architecture: A Review

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.

DNA Oxidation as Triggered by H3K9me2 Demethylation Drives Estrogen-Induced Gene Expression

Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomeraseIIbeta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.

Post-Translational Modifications of Nucleosomal Histones in Oligodendrocyte Lineage Cells in Development and Disease

The role of epigenetics in modulating gene expression in the development of organs and tissues and in disease states is becoming increasingly evident. Epigenetics refers to the several mechanisms modulating inheritable changes in gene expression that are independent of modifications of the primary DNA sequence and include post-translational modifications of nucleosomal histones, changes in DNA methylation, and the role of microRNA. This review focuses on the epigenetic regulation of gene expression in oligodendroglial lineage cells. The biological effects that post-translational modifications of critical residues in the N-terminal tails of nucleosomal histones have on oligodendroglial cells are reviewed, and the implications for disease and repair are critically discussed.

Nucleosomal Core Histones Mediate Dynamic Regulation of Poly(ADP-ribose) Polymerase 1 Protein Binding to Chromatin and Induction of Its Enzymatic Activity

Poly(ADP-ribose) polymerase 1 protein (PARP1) mediates chromatin loosening and activates the transcription of inducible genes, but the mechanism of PARP1 regulation in chromatin is poorly understood. We have found that PARP1 interaction with chromatin is dynamic and that PARP1 is exchanged continuously between chromatin and nucleoplasm, as well as between chromatin domains. Specifically, the PARP1 protein preferentially interacts with nucleosomal particles, and although the nucleosomal linker DNA is not necessary for this interaction, we have shown that the core histones, H3 and H4, are critical for PARP1 binding. We have also demonstrated that the histones H3 and H4 interact preferentially with the C-terminal portion of PARP1 protein and that the N-terminal domain of PARP1 negatively regulates these interactions. Finally, we have found that interaction with the N-terminal tail of the H4 histone triggers PARP1 enzymatic activity. Therefore, our data collectively suggests a model in which both the regulation of PARP1 protein binding to chromatin and the enzymatic activation of PARP1 protein depend on the dynamics of nucleosomal core histone mediation.

NAD+-dependent deacetylation of H4 lysine 16 by class III HDACs

Histone deacetylases (HDACs) catalyse the removal of acetyl groups from the N-terminal tails of histones. All known HDACs can be categorized into one of four classes (I-IV). The class III HDAC or silencing information regulator 2 (Sir2) family exhibits characteristics consistent with a distinctive role in regulation of chromatin structure. Accumulating data suggest that these deacetylases acquired new roles as genomic complexity increased, including deacetylation of non-histone proteins and functional diversification in mammals. However, the intrinsic regulation of chromatin structure in species as diverse as yeast and humans, underscores the pressure to conserve core functions of class III HDACs, which are also known as Sirtuins. One of the key factors that might have contributed to this preservation is the intimate relationship between some members of this group of proteins (SirT1, SirT2 and SirT3) and deacetylation of a specific residue in histone H4, lysine 16 (H4K16). Evidence accumulated over the years has uncovered a unique role for H4K16 in chromatin structure throughout eukaryotes. Here, we review the recent findings about the functional relationship between H4K16 and the Sir2 class of deacetylases and how that relationship might impact aging and diseases including cancer and diabetes.

Making binding relationships: Histone methylation

The nucleosome is the fundamental unit of chromatin and its highresolution Xray crystal struc ture was solved in 19971. This remarkable achievement showed the elegant and highly ordered packing of histones within 1.75 turns of DNA and it hinted at ways in which histones may be involved in regulating higherorder chromatin structure. Perhaps most striking is the way in which histone Nterminal tails protrude from their own nucleosome and, in some cases, make contact with adjacent nucleosomes. Simple extrapolation from this observation has implicated these interactions in higherorder chromatin regulation and many considered it likely that PTM (posttranslational modification) of the tails would affect internucleosomal stability and hence DNA processes such as transcription. Histone tails actually support multiple PTMs, including acetylation (see the article by Colyn CraneRobinson in this issue), phosphorylation and methylation. In this brief overview, I will consider histone methylation and discuss what we understand concerning its role.

Immediate‐early gene regulation by interplay between different post‐translational modifications on human histone H3

In mammalian cells, induction of immediate-early (IE) gene transcription occurs concomitantly with histone H3 phosphorylation on Ser 10 and is catalyzed by mitogen-activated protein kinases (MAPKs). Histone H3 is an evolutionarily conserved protein located in the core of the nucleosome, along with histones H2A, H2B, and H4. The N-terminal tails of histones protrude outside the chromatin structure and are accessible to various enzymes for post-translational modifications (PTM). Phosphorylation, O-GlcNAc modification, and their interplay often induce functional changes, but it is very difficult to monitor dynamic structural and functional changes in vivo. To get started in this complex task, computer-assisted studies are useful to predict the range in which those dynamic structural and functional changes may occur. As an illustration, we propose blocking of phosphorylation by O-GlcNAc modification on Ser 10, which may result in gene silencing in the presence of methylated Lys 9. Thus, alternate phosphorylation and O-GlcNAc modification on Ser 10 in the histone H3 protein may provide an on/off switch to regulate expression of IE genes.

The Fission Yeast Jmj2 Reverses Histone H3 Lysine 4 Trimethylation

Histone methylation regulates transcription, chromatin structure, and the epigenetic state of the cell. Recent studies identified the JmjC domain as a catalytic module for histone demethylation. Schizosaccharomyces pombe contains seven JmjC proteins, but it was unclear whether any of them functioned as histone demethylases. In this report, we show that the JmjC protein Jmj2, which is evolutionarily conserved from yeast to human, reversed trimethylated H3-Lys-4 to di- and mono-but not unmethylated products. Overexpression of Jmj2 but not a catalytically inactive mutant reduced H3-Lys-4 trimethylation levels in vivo and suppressed the toxicity caused by overexpression of the H3-Lys-4-me3-binding protein Yng1 in budding yeast. Genome-wide analysis showed that the loss of jmj2 was associated with an increase in the H3-Lys-4-me3 signal, which was enriched near the transcriptional start sites and the coding regions. At the mating-type locus, the loss of jmj2 or substitution of jmj2 with a catalytically inactive form is correlated with increased reporter gene transcription and H3-Lys-4-me3/2 levels, suggesting that Jmj2 and its demethylase activity may play a role in heterochromatin biology. Our findings identified a novel S. pombe histone demethylase with specificity toward di- and trimethylated histone H3-Lys-4 and a possible role in heterochromatin regulation.

Factors affecting the substrate specificity of histone deacetylases

Histone deacetylases (HDACs) catalyze the deacetylation of ε-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P -2-P -1-Lys(Ac)-MCA (P -1, P -2 = all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.