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The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF–SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.

Although a variety of affinity purification mass spectrometry (AP-MS) strategies have been used to investigate complex interactions, many of these are susceptible to artifacts because of substantial overexpression of the exogenously expressed bait protein. Here we present a logical and systematic workflow that uses the multifunctional Halo tag to assess the correct localization and behavior of tagged subunits of the Sin3 histone deacetylase complex prior to further AP-MS analysis. Using this workflow, we modified our tagging/expression strategy with 21.7% of the tagged bait proteins that we constructed, allowing us to quickly develop validated reagents. Specifically, we apply the workflow to map interactions between stably expressed versions of the Sin3 subunits SUDS3, SAP30, or SAP30L and other cellular proteins. Here we show that the SAP30 and SAP30L paralogues strongly associate with the core Sin3 complex, but SAP30L has unique associations with the proteasome and the myelin sheath. Next, we demonstrate an advancement of the complex NSAF (cNSAF) approach, in which normalization to the scaffold protein SIN3A accounts for variations in the proportion of each bait capturing Sin3 complexes and allows a comparison among different baits capturing the same protein complex. This analysis reveals that although the Sin3 subunit SUDS3 appears to be used in both SIN3A and SIN3B based complexes, the SAP30 subunit is not used in SIN3B based complexes. Intriguingly, we do not detect the Sin3 subunits SAP18 and SAP25 among the 128 high-confidence interactions identified, suggesting that these subunits may not be common to all versions of the Sin3 complex in human cells. This workflow provides the framework for building validated reagents to assemble quantitative interaction networks for chromatin remodeling complexes and provides novel insights into focused protein interaction networks.

Ochratoxin A (OTA) is a potent renal carcinogen but its mechanism has not been fully resolved. In vitro and in vivo gene expression studies consistently revealed down-regulation of gene expression as the predominant transcriptional response to OTA. Based on the importance of specific histone acetylation marks in regulating gene transcription and our recent finding that OTA inhibits histone acetyltransferases (HATs), leading to loss of acetylation of histones and non-histone proteins, we hypothesized that OTA-mediated repression of gene expression may be causally linked to HAT inhibition and loss of histone acetylation. In this study, we used a novel mass spectrometry approach employing chemical 13C-acetylation of unmodified lysine residues for quantification of post-translational acetylation sites to identify site-specific alterations in histone acetylation in human kidney epithelial cells (HK-2) exposed to OTA. These results showed OTA-mediated hypoacetylation at almost all lysine residues of core histones, including loss of acetylation at H3K9 and H3K14, which are hallmarks of gene activation. ChIP-qPCR used to establish a possible link between H3K9 or H3K14 hypoacetylation and OTA-mediated down-regulation of selected genes (AMIGO2, CLASP2, CTNND1) confirmed OTA-mediated H3K9 hypoacetylation at promoter regions of these genes. Integrated analysis of OTA-mediated genome-wide changes in H3K9 acetylation identified by ChIP-Seq with published gene expression data further demonstrated that among OTA-responsive genes almost 80% of hypoacetylated genes were down-regulated, thus confirming an association between H3K9 acetylation status and gene expression of these genes. However, only 7% of OTA repressed genes showed loss of H3K9 acetylation within promoter regions. Interestingly, however, GO analysis and functional enrichment of down-regulated genes showing loss of H3K9 acetylation at their respective promoter regions revealed enrichment of genes involved in the regulation of transcription, including a number of transcription factors that are predicted to directly or indirectly regulate the expression of 98% of OTA repressed genes. Thus, it is possible that histone acetylation changes in a fairly small set of genes but with key function in transcriptional regulation may trigger a cascade of events that may lead to overall repression of gene expression. Taken together, our data provide evidence for a mechanistic link between loss of H3K9 acetylation as a consequence of OTA-mediated inhibition of HATs and repression of gene expression by OTA, thereby affecting cellular processes critical to tumorigenesis.

Arsenic is a ubiquitous environmental contaminant with widespread public health concern. Epidemiological studies have revealed that chronic human exposure to arsenic in drinking water is associated with the prevalence of skin, lung, and bladder cancers. Aberrant histone modifications (e.g., methylation, acetylation, and ubiquitination) were previously found to be accompanied by arsenic exposure; thus, perturbation of epigenetic pathways is thought to contribute to arsenic carcinogenesis. Arsenite is known to interact with zinc finger motifs of proteins, and zinc finger motif is present in and indispensable for the enzymatic activities of crucial histone-modifying enzymes especially the MYST family of histone acetyltransferases (e.g., TIP60). Hence, we reasoned that trivalent arsenic may target the zinc finger motif of these enzymes, disturb their enzymatic activities, and alter histone acetylation. Herein, we found that As3+ could bind directly to the zinc-finger motif of TIP60 in vitro and in cells. In addition, exposure to As3+ could lead to a dose-dependent decrease in TIP60 protein level via the ubiquitin-proteasome pathway. Thus, the results from the present study revealed, for the first time, that arsenite may target cysteine residues in the zinc-finger motif of the TIP60 histone acetyltransferase, thereby altering the H4K16Ac histone epigenetic mark. Our results also shed some new light on the mechanisms underlying the arsenic-induced epigenotoxicity and carcinogenesis in humans.

Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.

γ-Aminobutyric acid A receptors (GABAA-Rs) mediate the majority of inhibitory neurotransmission in the adult brain. The α1-containing GABAA-Rs are the most prominent subtype in the adult brain and are important in both homeostatic function and several disease pathologies including alcohol dependence, epilepsy, and stress. Ethanol exposure causes a decrease of α1 transcription and peptide expression both in vivo and in vitro, but the mechanism that controls the transcriptional regulation is unknown. Because ethanol is known to activate epigenetic regulation of gene expression, we tested the hypothesis that ethanol regulates α1 expression through histone modifications in cerebral cortical cultured neurons. We found that class I histone deacetylases (HDACs) regulate ethanol-induced changes in α1 gene and protein expression as pharmacologic inhibition or knockdown of HDAC1-3 prevents the effects of ethanol exposure. Targeted histone acetylation associated with the Gabra1 promoter using CRISPR (clustered regularly interspaced palindromic repeat) dCas9-P300 (a nuclease-null Cas9 fused with a histone acetyltransferase) increases histone acetylation and prevents the decrease of Gabra1 expression. In contrast, there was no effect of a mutant histone acetyltransferase or generic transcriptional activator or targeting P300 to a distant exon. Conversely, using a dCas9-KRAB construct that increases repressive methylation (H3K9me3) does not interfere with ethanol-induced histone deacetylation. Overall our results indicate that ethanol deacetylates histones associated with the Gabra1 promoter through class I HDACs and that pharmacologic, genetic, or epigenetic intervention prevents decreases in α1 expression in cultured cortical neurons.

Phosphorylation of histone H3 affects transcription, chromatin condensation, and chromosome segregation. However, the role of phosphorylation of histone H2A remains unclear. Here, we found that Arabidopsis thaliana MUT9P-LIKE-KINASE (MLK4) phosphorylates histone H2A on serine 95, a plant-specific modification in the histone core domain. Mutations in MLK4 caused late flowering under long-day conditions but no notable phenotype under short days. MLK4 interacts with CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which allows MLK4 to bind to the GIGANTEA (GI) promoter. CCA1 interacts with YAF9a, a co-subunit of the Swi2/Snf2-related ATPase (SWR1) and NuA4 complexes, which are responsible for incorporating the histone variant H2A.Z into chromatin and histone H4 acetylase activity, respectively. Importantly, loss of MLK4 function led to delayed flowering by decreasing phosphorylation of H2A serine 95, along with attenuated accumulation of H2A.Z and the acetylation of H4 at GI, thus reducing GI expression. Together, our results provide insight into how phosphorylation of H2A serine 95 promotes flowering time and suggest that phosphorylation of H2A serine 95 modulated by MLK4 is required for the regulation of flowering time and is involved in deposition of the histone variant H2A.Z and H4 acetylation in Arabidopsis.

One of the main mechanisms of epigenetic control is post translational modification of histones, and one of the relatively less characterized, yet functionally important histone modifications is monoubiquitylation, which is reversed by histone deubiquitinases. In Arabidopsis, only two of such enzymes are known to date. One of them, OTLD1, deubiquitylates histone 2B and functions as a transcriptional repressor. But, could the same deubiquitinase act both as a repressor and an activator? Here, we addressed this question. Using gain-of-function and loss-of-function Arabidopsis alleles, we showed that OTLD1 can promote expression of a target gene. This transcriptional activation activity of OTLD1 involves occupation of the target chromatin by this enzyme, deubiquitination of monoubiquitylated H2B within the occupied regions, and formation of the euchromatic histone acetylation and methylation marks. Thus, OTLD1 can play a dual role in transcriptional repression and activation of its target genes. In these reactions, H2B ubiquitylation acts as both a repressive and an active mark whereas OTLD1 association with and deubiquitylation of the target chromatin may represent the key juncture between two opposing effects of this enzyme on gene expression.

Hox genes are responsible for encoding transcription factors that are essential for anterior-posterior body patterning at early stages of embryogenesis. However, detailed mechanisms of Hox genes are yet to be defined. Protein kinase B alpha (Akt1) was previously identified as a possible upstream regulator of Hox genes. Furthermore, the Hoxc11 gene has been upregulated in Akt1 null (Akt1-/-) mouse embryonic fibroblasts (MEFs), while repressed in wild-type MEFs. In this study, we propose to investigate the role of Gcn5, a histone acetyltransferase, in the regulation of Hoxc11 expression in MEFs. We showed that the H3 lysine 9 acetylation (H3K9ac) status has the same correlation with Hoxc11 expression and reported that Gcn5 is associated with the upregulation of Hoxc11 expression through H3K9ac in Akt1-/- MEFs. Since Hoxc11 was upregulated through histone acetylation in Akt1-/- MEFs, a functional role of Gcn5 on Hoxc11 expression was analyzed in Akt1-/- MEFs treated with Gcn5 specific inhibitor or transfected with Gcn5-small interfering RNA (Gcn5-siRNA). When the expression of Hoxc11 was analyzed using RT-PCR and real-time PCR, the Hoxc11 mRNA level was found to be similar in both Akt1-/- MEFs and control-siRNA transfected Akt1-/- MEFs. However, the Hoxc11 expression level was decreased in Gcn5-inhibited or Gcn5-knockdown Akt1-/- MEFs. Additionally, to analyze Gcn5-mediated histone acetylation status, chromatin immunoprecipitation assay was carried out in Gcn5-siRNA-transfected Akt1-/- MEFs. The H3K9ac at the Hoxc11 locus was decreased in Gcn5-knockdown Akt1-/- MEFs compared to controls. Based on these findings, we conclude that Gcn5 regulates Hoxc11 gene expression through mediating site-specific H3K9 acetylation in Akt1-/- MEFs.

Recent studies on enzymes and reader proteins for histone crotonylation support a function of histone crotonylation in transcription. However, the enzyme(s) responsible for histone decrotonylation (HDCR) remains poorly defined. Moreover, it remains to be determined if histone crotonylation is physiologically significant and functionally distinct from or redundant to histone acetylation. Here we present evidence that class I histone deacetylases (HDACs) rather than sirtuin family deacetylases (SIRTs) are the major histone decrotonylases, and that histone crotonylation is as dynamic as histone acetylation in mammalian cells. Notably, we have generated novel HDAC1 and HDAC3 mutants with impaired HDAC but intact HDCR activity. Using these mutants we demonstrate that selective HDCR in mammalian cells correlates with a broad transcriptional repression and diminished promoter association of crotonylation but not acetylation reader proteins. Furthermore, we show that histone crotonylation is enriched in and required for self-renewal of mouse embryonic stem cells.

Inhibitor of growth 2 (ING2) is involved in chromatin remodeling and it has previously been suggested that ING2 may regulate gene expression. The authors previously identified matrix metalloproteinase 13 (MMP13) as a target gene of ING2 in colorectal cancer. The aim of the present study was to identify novel genes regulated by ING2 and histone deacetylase 1 (HDAC1) and to clarify the biological significance of the ING2 structure. The present study generated the point mutant constructs of ING2 and deletion constructs consisting of partial ING2 to investigate the effect on gene expression and verify the interaction with HDAC1, mSin3A and sap30. A microarray was performed to find novel ING2/HDAC1 target genes using cell co-overexpression of ING2 and HDAC1. Plasminogen activator inhibitor-1 (PAI-1) was upregulated with overexpression of ING1b and ING2. The mutation of the PHD domain at 218 significantly attenuated the MMP13 and PAI-1 expression, whereas the mutation at 224 resulted in increased expression. Furthermore, the expression levels were slightly reduced by the mutation of the C-terminal. The lack of the PHD domain and the C-terminal in ING2 resulted in a decreased ability to induce gene expression. The C-terminal with PHD domain, which lacked the N-terminal, maintained the transactive function for regulating the target genes. In addition to MMP13 and PAI-1, eight genes [heat shock protein family A member 1A (HSPA1A), MIR7-3 host gene, chorionic somatomammotropin hormone 1, growth arrest and DNA damage inducible b, dehydrogenase/reductase 2, galectin 1, myosin light chain 1, and VGF nerve growth factor inducible] were demonstrated to be associated with ING2/HDAC1. The present study demonstrated that ING2/HDAC1 regulated PAI-1 and HSPA1A expression and the PHD domain and the C-terminal of ING2, which are binding sites of HDAC1 and mSin3A, are essential regions for the regulation of gene expression.

Dynamic changes in histone modifications under various physiological cues play important roles in gene transcription and cancer. Identification of new histone marks critical for cancer development is of particular importance. Here we show that, in a glucose-dependent manner, E3 ubiquitin ligase NEDD4 ubiquitinates histone H3 on lysine 23/36/37 residues, which specifically recruits histone acetyltransferase GCN5 for subsequent H3 acetylation. Genome-wide analysis of chromatin immunoprecipitation followed by sequencing reveals that NEDD4 regulates glucose-induced H3 K9 acetylation at transcription starting site and enhancer regions. Integrative analysis of ChIP-seq and microarray data sets also reveals a consistent role of NEDD4 in transcription activation and H3 K9 acetylation in response to glucose. Functionally, we show that NEDD4-mediated H3 ubiquitination, by transcriptionally activating IL1α, IL1β and GCLM, is important for tumour sphere formation. Together, our study reveals the mechanism for glucose-induced transcriptome reprograming and epigenetic regulation in cancer by inducing NEDD4-dependent H3 ubiquitination., Histone modifications play important roles in gene transcription and cancer. Here the authors establish a role for the E3 ubiquitin ligase NEDD4 in modifying in a glucose-dependent manner the histone H3, thus regulating the expression of genes involved in tumorigenesis.

Histone acetylation and deacetylation play an important role in plant growth and development by chromatin modifications. Regulation of histone acetylation and deacetylation is controlled by histone acetyltransferases and histone deacetylases (HDACs) in different tissues and development stages. Knowledge of the importance of genome stability, transcriptional regulation, development and response to stress has been obtained from the model plant Arabidopsis. However, little information on biological functions and development or stress-related HDAC genes is available in tomato. In this study, nine tomato histone deacetylase genes of the RPD3/HDA1 subfamily, from SlHDA1-SlHDA9, were characterized to encode histone deacetylase proteins that share high similarity of protein sequences and conserved domains with those of known plant HDACs. These HDAC genes have been further subdivided into four groups, namely Class I, Class II, Class III and Class IV based on phylogenetic analysis. Quantitative RT-PCR analysis revealed that the nine SlHDAC genes were expressed in all tissues with different transcript abundance and exhibited different tissue-specific expression profiles, suggesting that they may have crucial and diverse roles in tomato growth and development. Varying degrees of induction were detected in the transcript levels of these SlHDACs under various abiotic stresses including NaCl, dehydration, and high/low temperature. These nine SlHDACs were induced by the above stresses with differential/similar induction levels. Overall, this study provides valuable information for further exploring the regulation of HDAC genes during tomato development and fruit ripening and in response to environmental stresses.

Lipid-derived electrophiles (LDEs) directly modify proteins to modulate cellular signaling pathways in response to oxidative stress. One such LDE, 4-oxo-2-nonenal (4-ONE), has recently been found to target histones and interfere with histone assembly into nucleosomes. Unlike other LDEs that preferentially modify cysteine via nucleophilic Michael addition, 4-ONE reacts with histone lysine residues to form a new histone modification, gamma-oxononanoylation (Kgon). However, it remains unclear whether Kgon can cause irreversible damage or be regulated by enzymes "erasing" this nonenzymatic modification. Here, we report that human Sirt2 catalyzes the removal of histone Kgon. Among the tested human sirtuins, Sirt2 showed robust deacylase activity toward the Kgon-carrying histone peptides in vitro. We use alkynyl-4-ONE as a chemical reporter for Kgon to demonstrate that Sirt2 is responsible for removing histone Kgon in cells. Furthermore, we develop a ketone-reactive chemical probe to detect histones modified by endogenous 4-ONE in macrophages in response to inflammatory stimulation. Using this probe, we show Sirt2 as a deacylase able to control histone Kgon in stimulated macrophages. This study unravels a new mechanism for the regulation of LDE-derived protein posttranslational modifications, as well as a novel role played by Sirt2 as a histone Kgon deacylase in cytoprotective signaling responses.

Mammalian CXXC finger protein 1 (Cfp1) is a DNA-binding protein that is a component of the Setd1 histone methyltransferase complexes and is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a loss of histone H3-Lys4 tri-methylation (H3K4me3) at many CpG islands, and a mis-localization of this epigenetic mark to heterochromatic sub-nuclear domains. Furthermore, these cells fail to undergo cellular differentiation in vitro. These defects are rescued upon introduction of a Cfp1-expression vector. Cfp1 contains an N-terminal plant homeodomain (PHD), a motif frequently observed in chromatin associated proteins that functions as a reader module of histone marks. Here, we report that the Cfp1 PHD domain directly and specifically binds to histone H3K4me1/me2/me3 marks. Introduction of individual mutations at key Cfp1 PHD residues (Y28, D44, or W49) ablates this histone interaction both in vitro and in vivo. The W49A point mutation does not affect the ability of Cfp1 to rescue appropriate restriction of histone H3K4me3 to euchromatic sub-nuclear domains or in vitro cellular differentiation in Cfp1-null ES cells. Similarly, a mutated form of Cfp1 that lacks DNA-binding activity (C169A) rescues in vitro cellular differentiation. However, rescue of Cfp1-null ES cells with a double mutant form of Cfp1 (W49A, C169A) results in partially defective in vitro differentiation. These data define the Cfp1 PHD domain as a reader of histone H3K4me marks and provide evidence that this activity is involved in the regulation of lineage commitment in ES cells.

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP sequencing coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of noncanonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of proinflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition.

In the eukaryote nucleus, DNA wraps around histone cores and each core complex contains eight proteins including four pairs of H2A, H2B, H3, and H4 protein. The histone tail can be modified to alter the interaction strength between the histone core and a given DNA region, resulting in a change in gene expression. Various types of histone modifications are known to be involved in a number of cellular responses. In this study, we identified and characterized a novel mutant based on a growth assay using histone deacetylase inhibitors in combination with auxin, and we have named the mutant sensitive to histone deacetylase inhibitors 1. Our results imply that histone acetylation plays an important role in shoot morphology and leaf production in Arabidopsis.

Pygopus 2 (Pygo2) is a chromatin effector that plays an essential role in canonical Wnt signaling associated with development and stem cell growth. Its function is to facilitate histone acetylation by recruitment of histone acetyltransferases (HATs) at active sites of β-catenin-mediated transcription. In the present study, we report that Pygo2 itself is transiently acetylated when bound to the activated TCF/β-catenin transcription complex, which correlated with β-catenin binding and Axin2 gene activation. The HAT CBP/p300, but not GCN5/PCAF, targeted specific lysine residues of the N-terminal homology domain of Pygo2 for acetylation. Functional analyses revealed that the presence of CBP and p300 increased the association of Pygo2 with GCN5, independent of Pygo2 acetylation status. Finally, while acetylation of Pygo2 had little effect on active β-catenin complex formation, p300-mediated Pygo2 acetylation resulted in the displacement of Pygo2 from the nucleus to the cytoplasm by targeting specific lysine residues in the Pygo2 nuclear localization sequence. Taken together, these findings are consistent with a model in which acetylation of Pygo2 by CBP/p300 family members in the active TCF/β-catenin complex occurs coincident with histone acetylation and may be required for the recycling of Pygo2 away from the complex subsequent to target gene activation.

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442–29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

SummaryPHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first)-NSL (non-specific lethal) that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation readers in PHF20. We show that the plant homeodomain (PHD) finger of PHF20 recognizes dimethylated lysine 4 of histone H3 (H3K4me2) and represents an example of a native reader that selects for this modification. Biochemical and structural analyses help to explain this selectivity and the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a unique PHF20-mediated link between MOF histone acetyltransferase (HAT), p53, and H3K4me2, and suggest a model for rapid spreading of H4K16ac-enriched open chromatin.

Ethylene gas is essential for many developmental processes and stress responses in plants. EIN2 plays a key role in ethylene signalling but its function remains enigmatic. Here, we show that ethylene specifically elevates acetylation of histone H3K14 and the non-canonical acetylation of H3K23 in etiolated seedlings. The up-regulation of these two histone marks positively correlates with ethylene-regulated transcription activation, and the elevation requires EIN2. Both EIN2 and EIN3 interact with a SANT domain protein named EIN2 nuclear associated protein 1 (ENAP1), overexpression of which results in elevation of histone acetylation and enhanced ethylene-inducible gene expression in an EIN2-dependent manner. On the basis of these findings we propose a model where, in the presence of ethylene, the EIN2 C terminus contributes to downstream signalling via the elevation of acetylation at H3K14 and H3K23. ENAP1 may potentially mediate ethylene-induced histone acetylation via its interactions with EIN2 C terminus., The translocation of the C-terminal domain of EIN2 to the nucleus is essential for induction of gene expression in response to the plant hormone ethylene. Here, Zhang et al. show that EIN2 is required for ethylene-inducible elevation of histone acetylation marks associated with transcriptional activation.

Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein level as well as on the metabolome level. The acetyl group, donated by the metabolite acetyl-coenzyme A, can be co- or post-translationally attached to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. These reactions are catalyzed by various N-terminal and lysine acetyltransferases. In case of lysine acetylation, the reaction is enzymatically reversible via tightly regulated and metabolism-dependent mechanisms. The interplay between acetylation and deacetylation is crucial for many important cellular processes. In recent years, our understanding of protein acetylation has increased significantly by global proteomics analyses and in depth functional studies. This review gives a general overview of protein acetylation and the respective acetyltransferases, and focuses on the regulation of metabolic processes and physiological consequences that come along with protein acetylation.

The constitutively nuclear histone deacetylases (HDACs) 1, 2, and 3 erase acetyl marks on acetyllysine residues, alter the landscape of histone modifications, and modulate chromatin structure and dynamics and thereby crucially regulate gene transcription in higher eukaryotes. Nuclear HDACs exist as at least six giant multiprotein complexes whose nonenzymatic subunits confer genome targeting specificity for these enzymes. The deacetylase activity of HDACs has been shown previously to be enhanced by inositol phosphates, which also bridge the catalytic domain in protein–protein interactions with SANT (Swi3, Ada2, N-Cor, and TFIIIB) domains in all HDAC complexes except those that contain the Sin3 transcriptional corepressors. Here, using purified recombinant proteins, coimmunoprecipitation and HDAC assays, and pulldown and NMR experiments, we show that HDAC1/2 deacetylase activity in one of the most ancient and evolutionarily conserved Sin3L/Rpd3L complexes is inducibly up-regulated by inositol phosphates but involves interactions with a zinc finger motif in the Sin3-associated protein 30 (SAP30) subunit that is structurally unrelated to SANT domains, indicating convergent evolution at the functional level. This implies that this mode of regulation has evolved independently multiple times and provides an evolutionary advantage. We also found that constitutive association with another core subunit, Rb-binding protein 4 chromatin-binding factor (RBBP4), further enhances deacetylase activity, implying both inducible and constitutive regulatory mechanisms within the same HDAC complex. Our results indicate that inositol phosphates stimulate HDAC activity and that the SAP30 zinc finger motif performs roles similar to that of the unrelated SANT domain in promoting the SAP30–HDAC1 interaction and enhancing HDAC activity.

The circadian clock synchronizes endogenous rhythmic processes with environmental cycles and maximizes plant fitness. Multiple regulatory layers shape circadian oscillation, and chromatin modification is emerging as an important scheme for precise circadian waveforms. Here, we report the role of an evolutionarily conserved Sin3-histone deacetylase complex (HDAC) in circadian oscillation in Arabidopsis. SAP30 FUNCTION-RELATED 1 (AFR1) and AFR2, which are key components of Sin3-HDAC complex, are circadianly-regulated and possibly facilitate the temporal formation of the Arabidopsis Sin3-HDAC complex at dusk. The evening-expressed AFR proteins bind directly to the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and PSEUDO-RESPONSE REGULATOR 9 (PRR9) promoters and catalyze histone 3 (H3) deacetylation at the cognate regions to repress expression, allowing the declining phase of their expression at dusk. In support, the CCA1 and PRR9 genes were de-repressed around dusk in the afr1-1afr2-1 double mutant. These findings indicate that periodic histone deacetylation at the morning genes by the Sin3-HDAC complex contributes to robust circadian maintenance in higher plants.

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α-linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA-Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIβ as the targets of GCN5. Notably, the GCN5-dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild-type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering.

Summary Histone lysine acylations play an important role in the regulation of gene transcription in chromatin. Unlike histone acetyl-lysine, molecular recognition of a recently identified crotonyl-lysine mark is much less understood. Here, we report that the YEATS domain of AF9 preferentially binds crotonyl-lysine over acetyl-lysine in histone H3. Nuclear magnetic resonance structural analysis reveals that crotonyl-lysine of histone H3 lysine 18 is engulfed deep in an aromatic cage of the YEATS domain where the carbonyl oxygen of crotonyl-lysine forms a hydrogen bond with the backbone amide of protein residue Tyr78. The crotonyl-lysine, through its unique electron-rich double-bond side chain, engages π−π aromatic stacking and extended hydrophobic/aromatic interactions with the YEATS domain compared with acetyl-lysine. Our mutational analysis confirmed key protein residues Phe59 and Tyr78 for crotonyl-lysine recognition. Importantly, our findings present a new structural mechanism of protein-protein interactions mediated by histone lysine crotonylation, and show how the cells interpret acyl-lysine marks in different biological contexts.

Nucleosomes and modifications on the histones constitute the fundamental genomic structure of eukaryotes. It was reported that particular combinations of histone modifications occurring on the genome indicate specific chromatin states, which are related to a series of cell states. Although the histone code principle has been found for over a decade, the mechanism of the code is not yet known clearly. In this paper, we first conducted an extensive analysis on 38 histone modifications and 1 histone variant in human CD4+ T cells to reveal possible connections among these modifications. Then, we analyzed the different roles that histone modifications play in highly differentiated cells and undifferentiated cells, and found that the number of histone modifications on genes of differentiated cells is correlated to the expression levels of genes, while there is no direct effect of histone modification on gene expression level in undifferentiated cells. Finally, we compared the distributions of histone modifications of CD4+ T cell line and K562 cell line, and observed that they have similar distribution patterns. So we guess that there may exist certain conservative regions of histone modifications.

Histone proteins are essential for the organization, expression, and inheritance of genetic material for eukaryotic cells. A centromere-specific H3 histone variant, centromere protein A (CENP-A), shares about 50% amino acid sequence identity with H3. CENP-A is required for packaging the centromere and for the proper separation of chromosomes during mitosis. Despite their distinct biological functions, previously reported crystal structures of the CENP-A/H4 and H3/H4 dimers reveal a high degree of similarity. In this work, we characterize the structural dynamics of CENP-A/H4 and H3/H4 dimers based on a dual-resolution approach, using both microsecond-scale explicit-solvent all-atom and coarse-grained (CG) molecular dynamics (MD) simulations. Our data show that the H4 histone is significantly more rigid compared with the H3 histone and its variant CENP-A, hence, serving as a reinforcing structural element within the histone core. We report that the CENP-A/H4 dimer is significantly more dynamic than its canonical counterpart H3/H4, and our results provide a physical explanation for this flexibility. Further, we observe that the centromere-specific chaperone Holliday Junction Recognition Protein (HJURP) stabilizes the CENP-A/H4 dimer by forming a specific electrostatic interaction network. Finally, replacing CENP-A S68 with E68 disrupts the binding interface between CENP-A and HJURP in all-atom MD simulation, and consistently, in vivo experiments demonstrate that replacing CENP-A S68 with E68 disrupts CENP-A’s localization to the centromere. Based on all our results, we propose that, during the CENP-A/H4 deposition process, the chaperone HJURP protects various substructures of the dimer, serving both as a folding and binding chaperone.

Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating can function of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness in vitro and in vivo. The plant homeodomain (PHD) and Bromodomain cassette in ZMYND8 mediated the combinatorial recognition of H3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a previously unknown metastasis-suppressive epigenetic mechanism in which ZMYND8’s PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes.

The epigenome responds to changes in the extracellular environment, yet how this information is transmitted to the epigenetic regulatory machinery is unclear. Using a Saccharomyces cerevisiae yeast model, we demonstrate that target of rapamycin complex 1 (TORC1) signaling, which is activated by nitrogen metabolism and amino acid availability, promotes site-specific acetylation of histone H3 and H4 N-terminal tails by opposing the activity of the sirtuin deacetylases Hst3 and Hst4. TORC1 does so through suppression of the Tap42-regulated Sit4 (PP6) phosphatase complex, as sit4Δ rescues histone acetylation under TORC1-repressive conditions. We further demonstrate that TORC1 inhibition, and subsequent PP6 activation, causes a selective, rapid, nuclear accumulation of Hst4, which correlates with decreased histone acetylation. This increased Hst4 nuclear localization precedes an elevation in Hst4 protein expression, which is attributed to reduced protein turnover, suggesting that nutrient signaling through TORC1 may limit Hst4 nuclear accumulation to facilitate Hst4 degradation and maintain histone acetylation. This pathway is functionally relevant to TORC1 signaling since the stress sensitivity of a nonessential TORC1 mutant (tco89Δ) to hydroxyurea and arsenic can be reversed by combining tco89Δ with either hst3Δ, hst4Δ, or sit4Δ. Surprisingly, while hst3Δ or hst4Δ rescues the sensitivity tco89Δ has to low concentrations of the TORC1 inhibitor rapamycin, sit4Δ fails to do so. These results suggest Sit4 provides an additional function necessary for TORC1-dependent cell growth and proliferation. Collectively, this study defines a novel mechanism by which TORC1 suppresses a PP6-regulated sirtuin deacetylase pathway to couple nutrient signaling to epigenetic regulation.

Histones and polyamines are important determinants of the chromatin structure. Histones form the core of nucleosome particles and their modification by acetylation of N-terminal tails is involved in chromatin structural changes and transcriptional regulation. Polyamines, including spermidine, are also targets of both cytoplasmic and nuclear acetylation, which in turn alters their affinity for DNA and nucleosomes. Previous studies report the interplay between polyamines metabolism and levels of histone acetylation, but the molecular basis of this effect is still unclear. In this work, we have analyzed the in vitro effect of spermidine on histone H3 acetylation catalyzed by P/CAF, a highly conserved histone acetyltransferase (HAT) (E.C. 2.3.1.48). We have observed that spermidine at very low concentrations activates P/CAF, while it has an inhibitory effect at concentrations higher than 4 μM. In addition, the in vitro bimodal effect of spermidine on histone H3 acetylation was also distinctly observed in vivo on polytene chromosomes of Drosophila melanogaster. We also performed kinetic studies indicating that the activating effect of low spermidine concentrations on P/CAF-HAT activity is based on its involvement as a substrate for P/CAF to produce N

Concentrating factors in nuclear bodies is thought to promote efficient gene expression. Tatomer et al. show that the histone locus body (HLB) concentrates pre-mRNA processing factors at replication-dependent histone genes, resulting in optimal 3′ end formation of histone mRNAs coupled with transcription termination., The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination.

The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy-2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10-hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

Summary Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features., Graphical Abstract, Highlights • Active gene TSSs are marked by competing H4 K5K8 acetylation and butyrylation • Histone butyrylation directly stimulates transcription • H4K5 butyrylation prevents binding of the testis specific gene expression-driver Brdt • H4K5K8 butyrylation is associated with delayed histone removal in spermatogenic cells, Histone butyrylation stimulates gene transcription while competing with acetylation at H4K5 to control Brdt bromodomain binding. Differential chromatin labeling with interchangeable H4 acylations is an important epigenetic regulatory mechanism controlling gene expression and chromatin reorganization.

Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.

Here we report the identification and verification of a β-hydroxybutyrate-derived protein modification, lysine β-hydroxybutyrylation (Kbhb), as a new type of histone mark. Histone Kbhb marks are dramatically induced in response to elevated β-hydroxybutyrate levels in cultured cells, and in livers from mice subjected to prolonged fasting or streptozotocin-induced diabetic ketoacidosis. In total, we identified 44 histone Kbhb sites, a figure comparable to the known number of histone acetylation sites. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbhb is a mark enriched in active gene promoters, and that the increased H3K9bhb levels that occur during starvation are associated with genes up-regulated in starvation-responsive metabolic pathways. Histone β-hydroxybutyrylation thus represents a new epigenetic regulatory mark that couples metabolism to gene expression, offering a new avenue to study chromatin regulation and the diverse functions of β-hydroxybutyrate in the context of important human pathophysiological states, including diabetes, epilepsy, and neoplasia.

In metazoan the 3′-end processing of histone mRNAs is a conserved process involving the concerted action of many protein factors and the non-coding U7 snRNA. Recently, we identified that the processing of histone pre-mRNAs is promoted by an additional ncRNA, the Y3-derived Y3** RNA. U7 modulates the association of the U7 snRNP whereas Y3** promotes recruitment of CPSF (cleavage and polyadenylation specific factor) proteins to nascent histone transcripts at histone locus bodies (HLBs) in mammals. This enhances the 3′-end cleavage of nascent histone pre-mRNAs and modulates HLB assembly. Here we discuss new insights in the role of ncRNAs in the spatiotemporal control of histone synthesis. We propose that ncRNAs scaffold the formation of functional protein-RNA complexes and their sequential deposition on nascent histone pre-mRNAs at HLBs. These findings add to the multiple roles of ncRNAs in controlling gene expression and may provide new avenues for targeting histone synthesis in cancer.