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Somatic mutations affecting CREBBP and EP300 are a hallmark of diffuse large B-cell lymphoma (DLBCL). These mutations are frequently monoallelic, within the histone acetyltransferase (HAT) domain and usually mutually exclusive, suggesting that they might affect a common pathway, and their residual WT expression is required for cell survival. Using in vitro and in vivo models, we found that inhibition of CARM1 activity (CARM1i) slows DLBCL growth, and that the levels of sensitivity are positively correlated with the CREBBP/EP300 mutation load. Conversely, treatment of DLBCLs that do not have CREBBP/EP300 mutations with CARM1i and a CBP/p300 inhibitor revealed a strong synergistic effect. Our mechanistic data show that CARM1i further reduces the HAT activity of CBP genome wide and downregulates CBP-target genes in DLBCL cells, resulting in a synthetic lethality that leverages the mutational status of CREBBP/EP300 as a biomarker for the use of small-molecule inhibitors of CARM1 in DLBCL and other cancers.

Background: The mesoderm induction early response 1, family member 3 (MIER3) gene has been recognized as potentially being associated with cancer. However, in relation to the development of non-small cell lung cancer (NSCLC), the expression pattern and the role of MIER3 are yet to be reported. The aim of this research was to investigate the rate of expression of MIER3 in NSCLC cells and tissues and to investigate the role of MIER3 in NSCLC. Methods: Seventeen patients received NSCLC tissues and corresponding healthy tissues. MTT assay was used to determine cell proliferation. For detecting mRNA and protein expression, we used both quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot method. To measure cell apoptosis and cell cycle distribution, we applied the flow cytometry technique. We used a wound-healing assay and a Transwell invasion assay to study cell migration and invasion. Results: In comparison with adjacent normal tissues, the expression of MIER3 was down-regulated in NSCLC tissues. In addition, the level of MIER3 in NSCLC cell lines was also lower than in pulmonary epithelial cell BEAS-2B. Moreover, when MIER3 was overexpressed, cell proliferation, migration, and invasion were significantly inhibited, apoptosis increased, and cell cycle arrest was induced in A549 and H460 cells. MIER3 overexpression also suppressed tumor growth in NSCLC xenograft mouse models. Furthermore, our study demonstrated that MIER3 down-regulated the Wnt/β-catenin signaling pathway in NSCLC cells. More importantly, MIER3 decreased the activity of histone acetyltransferase (HAT) p300, which may have contributed to its regulation on β-catenin and tumorigenesis. Conclusions: The data suggests MIER3 takes on the tumor-suppressor role in the progression of NSCLC and, therefore, could prove to be a valuable clinical marker in the prognosis of the disease.

Histone acetyltransferase (HAT) activity is well established to regulate inflammatory responses. In contrast, the mechanisms by which natural nutritional extracts influence epigenetic mechanisms to regulate inflammation have not yet been thoroughly investigated. Thus, in the present study, we observed that the anti-HAT activity exerted by an ethanol extract of Ligularia fischeri (ELF) inhibited inflammation. Specifically, we used a cell-free system to show that ELF attenuates HAT activity. We also demonstrated that ELF decreases lipopolysaccharide (LPS)-induced HAT mRNA and protein expression levels in Raw 264.7 cells, and thereby attenuates inflammation-induced patterns of hyperacetylation at nonhistone and histone-H4 proteins. Interestingly, we found that ELF blocked p65 translocation in LPS-stimulated Raw 264.7 cells by attenuating acetylation at lysine residue 310 of p65. Finally, we investigated whether ELF reduces the inflammatory cytokines, IL-6, IL-1β, and TNFα, using its HAT inhibitor activity. Taken together, these results suggest that ELF negatively regulates inflammatory responses by inhibiting HATs and HAT activity.

A novel mushroom-like electrochemical immunoassay for the ultrasensitive detection of histone acetyltransferase activity (HAT p300) has been established on account of the new composite graphene oxide (GO) nanolayer. The immunoassay involves immobilization of substrate peptide onto Au electrode, acetylation in lysine of substrate peptide, and the interaction between acetyl group of lysine and acetyl-antibody (AbAc) of the GO nanolayer. The GO nanolayer comprises large amounts of methylene blue molecules (MB), giving rise to large signal amplification. Only in the presence of HAT p300, an obvious electrochemical signal appears and the peak linear current is proportion to the HAT p300 concentrations ranging from 0.01 to 150 nM with a detection limit of 0.0036 nM. The great enhancement on sensitivity of the proposed mushroom-like immunosensor derives from both the constructed Faraday cage and the extended outer Helmholtz plane (OHP). Further, the immunosensor with excellent sensitivity and selectivity can be applied for the HAT p300 activity detection in Hela cell lysates, serum and urine, hinting an improved and splendid analytical performance. Briefly, this stable, simple and ultrasensitive electrochemical immunoassay has considerable promise for further applications in the HATs-interrelated epigenetic studies and drug development.

Acetylation of lysine in the histone H4 N-terminal is one of the most significant epigenetic modifications in cells. Aberrant changes involving lysine acetylation modification are commonly reported in multiple types of cancers. Currently, whether it is for in vivo or in vitro, there are limited approaches for the detection of H4 lysine acetylation levels. In particular, the main problems are the high cost and the cumbersome detection process, such as for radioactive 14C isotope detection. Therefore, there is an important need to develop a simple, fast, and low-cost means of detection. In this study, we reported the development of a gene-coding protein sensor. This protein sensor was designed based on the mechanism of fluorescence resonance energy transfer (FRET). The four kinds of sensors, varying from substrate and linker length, were evaluated, with ~20% increases in response efficiency. Next, sensors with different lysine mutation sites in the substrate sequence or mutation of key amino acids in the binding domain were also analyzed to determine site specificity. We found single-site lysine mutant could not cause a significant decrease in response efficiency. Furthermore, addition of MG149, a histone acetyltransferase inhibitor, resulted in a decrease in the ratio change value. Moreover, histone deacetylase1 HDAC1 was also found to reduce the ratio change values when added to reaction system. Finally, the optimized sensor was applied to living cells and established to provide a sensitive response with overexpression and knockdown of MOF (males absent on the first). These results indicated that the sensor can be used for screening chemical drugs regulating H4 N-terminal lysine acetylation level in vitro, as well as monitoring dynamic changes of lysine acetylation levels in living cells.

Summary Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.

One of the major barriers in the prevention and control of malaria programs worldwide is the growing emergence of multidrug resistance in Plasmodium parasites, and this necessitates continued efforts to discover and develop effective drug molecules targeting novel proteins essential for parasite survival. In recent years, epigenetic regulators have evolved as an attractive drug target option owing to their crucial role in survival and development of Plasmodium at different stages of its life cycle. PfMYST, a histone acetyltransferase protein, is known to regulate key cellular processes, such as cell cycle progression, DNA damage repair, and antigenic variation, that facilitate parasite growth, adaptation, and survival inside its host. With the aim of assessing the therapeutic potential of PfMYST as a novel drug target, we examined the effect of NU9056 (an HsTIP60 inhibitor) on the rate of parasite growth and survival. In the present study, by using a yeast complementation assay, we established that PfMYST is a true homolog of TIP60 and showed that NU9056 can inhibit PfMYST catalytic activity and kill P. falciparum parasites in culture. Inhibiting the catalytic activity of PfMYST arrests the parasite in the trophozoite stage and inhibits its further transition to the schizont stage, eventually leading to its death. Overall, our study provides proof of concept that PfMYST catalytic activity is essential for parasite growth and survival and that PfMYST can be a potential target for antimalarial therapy.

Targeting epigenetics in cancer has emerged as a promising anticancer strategy. p300/CBP is a central regulator of epigenetics and plays an important role in hepatocellular carcinoma (HCC) progression. Tumor-associated metabolic alterations contribute to the establishment and maintenance of the tumorigenic state. In this study, we used a novel p300 inhibitor, B029-2, to investigate the effect of targeting p300/CBP in HCC and tumor metabolism. p300/CBP–mediated acetylation of H3K18 and H3K27 increased in HCC tissues compared with surrounding noncancerous tissues. Conversely, treatment with B029-2 specifically decreased H3K18Ac and H3K27Ac and displayed significant antitumor effects in HCC cells in vitro and in vivo. Importantly, ATAC-seq and RNA-seq integrated analysis revealed that B029-2 disturbed metabolic reprogramming in HCC cells. Moreover, B029-2 decreased glycolytic function and nucleotide synthesis in Huh7 cells by reducing H3K18Ac and H3K27Ac levels at the promoter regions of amino acid metabolism and nucleotide synthesis enzyme genes, including PSPH, PSAT1, ALDH18A1, TALDO1, ATIC, and DTYMK. Overexpression of PSPH and DTYMK partially reversed the inhibitory effect of B029-2 on HCC cells. These findings suggested that p300/CBP epigenetically regulates the expression of glycolysis-related metabolic enzymes through modulation of histone acetylation in HCC and highlights the value of targeting the histone acetyltransferase activity of p300/CBP for HCC therapy. Significance: This study demonstrates p300/CBP as a critical epigenetic regulator of glycolysis-related metabolic enzymes in HCC and identifies the p300/CBP inhibitor B029-2 as a potential therapeutic strategy in this disease.

The transition from multipotent neural stem cells (NSCs) to terminally differentiated neurons is a multistep process, and the transition is finely regulated by transcription factors with basic helix-loop-helix (bHLH) motifs. Melatonin is an endogenous neurohormone with profound neurotrophic and neuroprotective effects both during the embryonic developmental stage and adulthood. The effects of melatonin on the differentiation of NSCs have been reported, and these effects may be responsible for its neuroprotective properties. However, the mechanisms underlying the effects of melatonin are not well understood. It is unclear whether melatonin affects the expression of bHLH factors at the onset of neuronal differentiation, and the molecular mechanisms involved still need to be further explored. Using mouse NSCs, we identified a novel role for melatonin in the epigenetic regulation of bHLH factors during neuronal differentiation. Our data showed that melatonin promoted neuronal differentiation by specifically increasing the acetylation of histone H3 lysine14 (H3K14). Increased H3K14 acetylation altered the chromatin state of the promoters of bHLH factors Neurogenin1 and NeuroD1 and activated their transcription; then, Neurogenin1 and NeuroD1 initiated and sustained the commitment to neuronal fates. As we know, CBP/p300 is an important class of histone acetyltransferases that acetylate histone H3K14, we found that melatonin activated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300 via ERK signaling pathways. For the first time, we systematically showed the molecular mechanism of action of melatonin, which suggested that melatonin functions as a regulator of the acetylation-dependent gene expression network.

The human males absent on the first (MOF)-containing histone acetyltransferase nonspecific lethal (NSL) complex comprises nine subunits including the O-linked N-acetylglucosamine (O-GlcNAc) transferase, isoform 1 (OGT1). However, whether the O-GlcNAc transferase activity of OGT1 controls histone acetyltransferase activity of the NSL complex and whether OGT1 physically interacts with the other NSL complex subunits remain unclear. Here, we demonstrate that OGT1 regulates the activity of the NSL complex by mainly acetylating histone H4 Lys-16, Lys-5, and Lys-8 via O-GlcNAcylation and stabilization of the NSL complex subunit NSL3. Knocking down or overexpressing OGT1 in human cells remarkably affected the global acetylation of histone H4 residues Lys-16, Lys-5, and Lys-8. Because OGT1 is a subunit of the NSL complex, we also investigated the function of OGT1 in this complex. Co-transfection/co-immunoprecipitation experiments combined with in vitro O-GlcNAc transferase assays confirmed that OGT1 specifically binds to and O-GlcNAcylates NSL3. In addition, wheat germ agglutinin affinity purification verified the occurrence of O-GlcNAc modification on NSL3 in cells. Moreover, O-GlcNAcylation of NSL3 by wild-type OGT1 (OGT1-WT) stabilized NSL3. This stabilization was lost after co-transfection of NSL3 with an OGT1 mutant, OGT1C964A, that lacks O-GlcNAc transferase activity. Furthermore, stabilization of NSL3 by OGT1-WT significantly increased the global acetylation levels of H4 Lys-5, Lys-8, and Lys-16 in cells. These results suggest that OGT1 regulates the activity of the NSL complex by stabilizing NSL3.

Histone lysine acetylation is thought to play a role in regulating the balance between energy storage and energy expenditure. However, the epigenetic mechanisms by which food phytochemicals influence metabolic processes in the liver have not been thoroughly investigated. In this study, we investigated the effect of an ethanol extract of Capsella bursa-pastoris (ECB) on histone acetyltransferase (HAT) inhibition, and whether it could thereby attenuate lipid accumulation in vitro and in vivo. We observed that ECB inhibits HAT activity as assessed by colorimetric and autoradiography assay systems. ECB also reduced oleic acid (OA)-stimulated histone acetylation at H4K5 and H4K12 and attenuated OA-mediated lipid accumulation in HepG2 cells, in the absence of observable cytotoxicity. We then investigated these effects in vivo. Mice were fed on either a normal diet (ND) or high-fat diet (HFD) in the presence or absence of ECB supplementation. In comparison with the ND controls, the HFD mice exhibited higher body weight, liver fat, adipose tissue size, and total serum cholesterol concentrations, and these effects were significantly attenuated by ECB supplementation. Taken together, these results suggest that ECB protects against the mechanisms responsible for HFD-induced hepatic steatosis, and may involve the targeting of histone H4K acetylation.

HBO1, a member of the MYST family of histone acetyltransferases (HATs), is required for global acetylation of histone H3K14 and embryonic development. It functions as a catalytic subunit in multisubunit complexes comprising a BRPF1/2/3 or JADE1/2/3 scaffold protein, and two accessory proteins. BRPF2 has been shown to be important for the HAT activity of HBO1 toward H3K14. Here we demonstrated that BRPF2 can regulate the HAT activity of HBO1 toward free H3 and H4, and nucleosomal H3. Particularly, a short N-terminal region of BRPF2 is sufficient for binding to HBO1 and can potentiate its activity toward H3K14. The crystal structure of the HBO1 MYST domain in complex with this segment of BRPF2 together with the biochemical and cell biological data revealed the key residues responsible for the HBO1–BRPF2 interaction. Our structural and functional data together indicate that the N-terminal region of BRPF2 plays an important role in the binding of HBO1 and a minor role in the binding of nucleosomes, which provide new mechanistic insights into the regulation of the HAT activity of HBO1 by BRPF2.

Chemical inhibition of chromatin regulators provides an effective approach to investigate the roles of chromatin modifications in plant and animals. In this work, chemical inhibition of the Arabidopsis histone acetyltransferase activity by γ-butyrolactone (MB-3), the inhibitor of the catalytic activity of mammalian GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is evaluated. Arabidopsis seedlings were germinated in LS medium supplemented with different concentrations of MB-3, and inhibition in the root length and yellowed leaves were observed. The yellowed leaves phenotype of the plants grown in 100 μM of MB-3 was reverted when plants were additionally treated with 1 μM of TSA, a histone deacetylase inhibitor. Using an immunoblot assay with specific antibodies revealed a reduction of H3K14 acetylation levels at 3 and 24 h post-treatment. At 24 h post-treatment a reduction of H3K9 acetylation levels was observed. Targets of GCN5 related to stress were downregulated at 3 h post-treatment but no change was observed in target genes related to developmental transition. Our results indicate that MB-3 is a chemical inhibitor of the histone acetyltransferase in Arabidopsis and suggest that this inhibitor could function in other plants species.

Histone post-translational modifications are essential for the regulation of gene expression in eukaryotes. Gcn5 (KAT2A) is a histone acetyltransferase that catalyzes the post-translational modification at multiple positions of histone H3 through the transfer of acetyl groups to the free amino group of lysine residues. Gcn5 catalyzes histone acetylation in the context of a HAT module containing the Ada2, Ada3 and Sgf29 subunits of the parent megadalton SAGA transcriptional coactivator complex. Biochemical and structural studies have elucidated mechanisms for Gcn5’s acetyl- and other acyltransferase activities on histone substrates, for histone H3 phosphorylation and histone H3 methylation crosstalks with histone H3 acetylation, and for how Ada2 increases Gcn5’s histone acetyltransferase activity. Other studies have identified Ada2 isoforms in SAGA-related complexes and characterized variant Gcn5 HAT modules containing these Ada2 isoforms. In this review, we highlight biochemical and structural studies of Gcn5 and its functional interactions with Ada2, Ada3 and Sgf29.

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

// Seung-Hee Ryu 1 , Eun-Young Park 1 , Sungmin Kwak 3, 4 , Seung-Ho Heo 2 , Je-Won Ryu 2 , Jin-hong Park 1 , Kyung-Chul Choi 3, 4, * , Sang-wook Lee 1, * 1 Department of Radiation Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Institute for Life Sciences, Asan Medical Center, Seoul, Korea 3 Department of Biomedical Sciences and Department of Pharmacology, Cell Dysfunction Research Center (CDRC), University of Ulsan College of Medicine, Seoul, Korea 4 Cell Dysfunction Research Center and Bio-Medical Institute of Technology (BMIT), University of Ulsan College of Medicine, Seoul, Korea * These authors contributed equally to this work Correspondence to: Sang-wook Lee, e-mail: lsw@amc.seoul.kr Kyung-Chul Choi, e-mail: choikc75@amc.seoul.kr Keywords: radiation-induced fibrosis, α-lipoic acid, acetylation, NF-κB, PAI-1 Received: July 07, 2015 Accepted: December 05, 2015 Published: January 20, 2016 ABSTRACT Radiation-induced fibrosis (RIF) is one of the most common late complications of radiation therapy. We found that α-lipoic acid (α-LA) effectively prevents RIF. In RIF a mouse model, leg contracture assay was used to test the in vivo efficacy of α-LA. α-LA suppressed the expression of pro-fibrotic genes after irradiation, both in vivo and in vitro , and inhibited the up-regulation of TGF-β1-mediated p300/CBP activity. Thus, α-LA prevents radiation-induced fibrosis (RIF) by inhibiting the transcriptional activity of NF-κB through inhibition of histone acetyltransferase activity. α-LA is a new therapeutic methods that can be used in the prevention-treatment of RIF.

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.

Histone acetylation is crucial transcription-activating modification, which is achieved by histone acetyltransferase using acetyl coenzyme A as acetyl donor. As by-product, the concentration of coenzyme A (CoA) is related to histone acetyltransferase. Based on it, a newly and sensitively photoelectrochemical method was designed for histone acetyltransferase detection, where WS2 nanosheets were employed as photoactive material, polydopamine (PDA) was used as solid-state electron donor, SMCC was adopted as crosslinker for capture CoA through the covalent reaction between SMCC and –SH of CoA, and streptavidin was used as signal inhibition unit. This novel PEC strategy presented a superior linear relationship with the range of 0.1–200 nM. The detection limit was 47 pM (S/N = 3). This assay was further applied to investigate the effect of heavy metal and anacardic acid on the effect of histone acetyltransferase activity, provided a platform for the screening of histone acetyltransferase inhibitor, and assessing the applicability of this method in ecotoxicological effect evaluation.

This study shows that genetic or pharmacological manipulation of the TCA cycle, when mitochondria are dysfunctional, can modulate histone acetylation and gene expression in the nucleus with physiological outcomes., The impact of mitochondrial dysfunction in epigenetics is emerging, but our understanding of this relationship and its effect on gene expression remains incomplete. We previously showed that acute mitochondrial DNA (mtDNA) loss leads to histone hypoacetylation. It remains to be defined if these changes are maintained when mitochondrial dysfunction is chronic and if they alter gene expression. To fill these gaps of knowledge, we here studied a progressive and a chronic model of mtDNA depletion using biochemical, pharmacological, genomics, and genetic assays. We show that histones are primarily hypoacetylated in both models. We link these effects to decreased histone acetyltransferase activity unrelated to changes in ATP citrate lyase, acetyl coenzyme A synthetase 2, or pyruvate dehydrogenase activities, which can be reversibly modulated by altering the mitochondrial pool of acetyl-coenzyme A. Also, we determined that the accompanying changes in histone acetylation regulate locus-specific gene expression and physiological outcomes, including the production of prostaglandins. These results may be relevant to the pathophysiology of mtDNA depletion syndromes and to understanding the effects of environmental agents that lead to physical or functional mtDNA loss.

Integrase strand transfer inhibitors (INSTIs) are the newest class of antiretrovirals to be approved for the treatment of HIV infection. Canonical resistance to these competitive inhibitors develops through substitutions in the integrase active site that disrupt drug-protein interactions. However, resistance against the newest integrase inhibitor, dolutegravir (DTG), is associated with an R263K substitution at the C terminus of integrase that causes resistance through an unknown mechanism. The integrase C-terminal domain is involved in many processes over the course of infection and is posttranslationally modified via acetylation of three lysine residues that are important for enzyme activity, integrase multimerization, and protein-protein interactions. Here we report that regulation of the acetylation of integrase is integral to the replication of HIV in the presence of DTG and that the R263K mutation specifically disrupts this regulation, likely due to enhancement of interactions with the histone deacetylase I complex, as suggested by coimmunoprecipitation assays. Although no detectable differences in the levels of cell-free acetylation of the wild-type (WT) and mutated R263K enzymes were observed, the inhibition of cellular histone acetyltransferase enzymes sensitized the NL4.3

Accumulating findings demonstrate the importance of histone acetyltransferases (HATs) in regulating the acetylation of histones and reveal that their aberrant catalytic activities are involved in the occurrence and progress of numerous diseases. Herein, a feasible electrochemical method is proposed to assay the activity of HAT. The critical elements of the assay method are the hindrance of HAT-catalyzed acetylation against carboxypeptidase Y-catalyzed digestion and cucurbit[8]uril-assisted peptide assembly, which may recruit peptide-templated silver nanoparticles onto the electrode surface, producing significant electrochemical signals. Taking p300 as a model HAT, the assay method is validated to exhibit desirable selectivity, reproducibility, and usability in inhibitor analysis, and allow absolute activity determination in a linear range from 0.1 to 50 nM with a detection limit of 0.055 nM, which is lower than those of previous reports. Therefore, this work may provide an effective tool for HAT activity assay, which will be of great potential in HAT-related fundamental research, disease diagnosis, and drug development in the future. Graphical abstract ᅟ.

Chronic myelomonocytic leukemia (CMML) constitutes a hematopoietic stem cell (HSC) disorder characterized by prominent monocytosis and myelodysplasia. Although genome sequencing has revealed the CMML mutation profile, the mechanism of disease development remains unclear. Here we show that aberrant histone acetylation by nucleoporin-98 (NUP98)-HBO1, a newly identified fusion in a patient with CMML, is sufficient to generate clinically relevant CMML pathogenesis. Overexpression of NUP98-HBO1 in murine HSC/progenitors (HSC/Ps) induced diverse CMML phenotypes, such as severe leukocytosis, increased CD115+ Ly6Chigh monocytes (an equivalent subpopulation to human classical CD14+ CD16− monocytes), macrocytic anemia, thrombocytopenia, megakaryocyte-lineage dysplasia, splenomegaly, and cachexia. A NUP98-HBO1–mediated transcriptional signature in human CD34+ cells was specifically activated in HSC/Ps from a CMML patient cohort. Besides critical determinants of monocytic cell fate choice in HSC/Ps, an oncogenic HOXA9 signature was significantly activated by NUP98-HBO1 fusion through aberrant histone acetylation. Increased HOXA9 gene expression level with disease progression was confirmed in our CMML cohort. Genetic disruption of NUP98-HBO1 histone acetyltransferase activity abrogated its leukemogenic potential and disease development in human cells and a mouse model. Furthermore, treatment of azacytidine was effective in our CMML mice. The recapitulation of CMML clinical phenotypes and gene expression profile by the HBO1 fusion suggests our new model as a useful platform for elucidating the central downstream mediators underlying diverse CMML-related mutations and testing multiple compounds, providing novel therapeutic potential.

Phenotypic plasticity is common in development. Candida albicans, a polymorphic fungal pathogen of humans, possesses the unique ability to achieve rapid and reversible cell fate between unicellular form (yeast) and multicellular form (hypha) in response to environmental cues. The NuA4 histone acetyltransferase activity and Hda1 histone deacetylase activity have been reported to be required for hyphal initiation and maintenance. However, how Hda1 and NuA4 regulate hyphal elongation is not clear. NuA4 histone acetyltransferase and SWR1 chromatin remodeling complexes are conserved from yeast to human, which may have merged together to form a larger TIP60 complex since the origin of metazoan. In this study, we show a dynamic merge and separation of NuA4 and SWR1 complexes in C. albicans. NuA4 and SWR1 merge together in yeast state and separate into two distinct complexes in hyphal state. We demonstrate that acetylation of Eaf1 K173 controls the interaction between the two complexes. The YEATS domain of Yaf9 in C. albicans can recognize an acetyl-lysine of the Eaf1 and mediate the Yaf9-Eaf1 interaction. The reversible acetylation and deacetylation of Eaf1 by Esa1 and Hda1 control the merge and separation of NuA4 and SWR1, and this regulation is triggered by Brg1 recruitment of Hda1 to chromatin in response nutritional signals that sustain hyphal elongation. We have also observed an orchestrated promoter association of Esa1, Hda1, Swr1, and H2A.Z during the reversible yeast–hyphae transitions. This is the first discovery of a regulated merge of the NuA4 and SWR1 complexes that controls cell fate determination and this regulation may be conserved in polymorphic fungi.

The heparan sulfate proteoglycan syndecan-1 is proteolytically shed from the surface of multiple myeloma cells and is abundant in the bone marrow microenvironment where it promotes tumor growth, angiogenesis, and metastasis. In this study, we demonstrate for the first time that shed syndecan-1 present in the medium conditioned by tumor cells is taken up by bone marrow-derived stromal cells and transported to the nucleus. Translocation of shed syndecan-1 (sSDC1) to the nucleus was blocked by addition of exogenous heparin or heparan sulfate, pretreatment of conditioned medium with heparinase III, or growth of cells in sodium chlorate, indicating that sulfated heparan sulfate chains are required for nuclear translocation. Interestingly, cargo bound to sSDC1 heparan sulfate chains (i.e. hepatocyte growth factor) was transported to the nucleus along with sSDC1, and removal of heparan sulfate-bound cargo from sSDC1 abolished its translocation to the nucleus. Once in the nucleus, sSDC1 binds to the histone acetyltransferase enzyme p300, and histone acetyltransferase activity and histone acetylation are diminished. These findings reveal a novel function for shed syndecan-1 in mediating tumor-host cross-talk by shuttling growth factors to the nucleus and by altering histone acetylation in host cells. In addition, this work has broad implications beyond myeloma because shed syndecan-1 is present in high levels in many tumor types as well as in other disease states.

Background The major human fungal pathogen Candida albicans possesses a diploid genome, but responds to growth in challenging environments by employing chromosome aneuploidy as an adaptation mechanism. For example, we have shown that C. albicans adapts to growth on the toxic sugar l-sorbose by transitioning to a state in which one chromosome (chromosome 5, Ch5) becomes monosomic. Moreover, analysis showed that while expression of many genes on the monosomic Ch5 is altered in accordance with the chromosome ploidy, expression of a large fraction of genes is increased to the normal diploid level, presumably compensating for gene dose. Results In order to understand the mechanism of the apparent dosage compensation, we now report genome-wide ChIP-microarray assays for a sorbose-resistant strain harboring a monosomic Ch5. These data show a significant chromosome-wide elevation in histone H4 acetylation on the mCh5, but not on any other chromosome. Importantly, strains lacking subunits of the NuA4 H4 histone acetyltransferase complex, orthologous to a complex previously shown in Drosophila to be associated with a similar gene dosage compensation mechanism, did not show an increase in H4 acetylation. Moreover, loss of NuA4 subunits severely compromised the adaptation to growth on sorbose. Conclusions Our results are consistent with a model wherein chromosome-wide elevation of H4 acetylation mediated by the NuA4 complex plays a role in increasing gene expression in compensation for gene dose and adaption to growth in a toxic environment.

Integrase strand transfer inhibitors (INSTIs) are the newest class of antiretrovirals to be approved for the treatment of HIV infection. Canonical resistance to these competitive inhibitors develops through substitutions in the integrase active site that disrupt drug-protein interactions. However, resistance against the newest integrase inhibitor, dolutegravir (DTG), is associated with an R263K substitution at the C terminus of integrase that causes resistance through an unknown mechanism. The integrase C-terminal domain is involved in many processes over the course of infection and is posttranslationally modified via acetylation of three lysine residues that are important for enzyme activity, integrase multimerization, and protein-protein interactions. Here we report that regulation of the acetylation of integrase is integral to the replication of HIV in the presence of DTG and that the R263K mutation specifically disrupts this regulation, likely due to enhancement of interactions with the histone deacetylase I complex, as suggested by coimmunoprecipitation assays. Although no detectable differences in the levels of cell-free acetylation of the wild-type (WT) and mutated R263K enzymes were observed, the inhibition of cellular histone acetyltransferase enzymes sensitized the NL4.3 WT virus to DTG, while NL4.3 R263K was almost completely unaffected. When levels of endogenous acetylation were manipulated in virus-producing cells, inhibitors of acetylation enhanced the replication of NL4.3 R263K , whereas inhibition of deacetylation greatly diminished the replication of NL4.3 WT . Taken together, these results point to a pivotal role of acetylation in the resistance mechanism of HIV to some second-generation integrase strand transfer inhibitors, such as DTG. IMPORTANCE This is, to our knowledge, the first report of the influence of posttranslational modifications on HIV drug resistance. Both viral replication and resistance to second-generation integrase strand transfer inhibitors of both WT and INSTI-resistant HIV strains were differentially affected by acetylation, likely as a result of altered interactions between integrase and the cellular deacetylation machinery. Many “shock and kill” strategies to eradicate HIV manipulate endogenous levels of acetylation in order to reactivate latent HIV. However, our results suggest that some drug-resistant viruses may differentially respond to such stimulation, which may complicate the attainment of this goal. Our future work will further illuminate the mechanisms involved.

Rubinstein-Taybi syndrome (RSTS) is a rare genetic disorder in humans characterized by growth and psychomotor delay, abnormal gross anatomy, and mild to severe mental retardation (Rubinstein and Taybi, Am J Dis Child 105:588–608, 1963, Hennekam et al., Am J Med Genet Suppl 6:56–64, 1990). RSTS is caused by de novo mutations in epigenetics-associated genes, including the cAMP response element-binding protein (CREBBP), the gene-encoding protein referred to as CBP, and the EP300 gene, which encodes the p300 protein, a CBP homologue. Recent studies of the epigenetic mechanisms underlying cognitive functions in mice provide direct evidence for the involvement of nuclear factors (e.g., CBP) in the control of higher cognitive functions. In fact, a role for CBP in higher cognitive function is suggested by the finding that RSTS is caused by heterozygous mutations at the CBP locus (Petrij et al., Nature 376:348–351, 1995). CBP was demonstrated to possess an intrinsic histone acetyltransferase activity (Ogryzko et al., Cell 87:953–959, 1996) that is required for CREB-mediated gene expression (Korzus et al., Science 279:703–707, 1998). The intrinsic protein acetyltransferase activity in CBP might directly destabilize promoter-bound nucleosomes, facilitating the activation of transcription. Due to the complexity of developmental abnormalities and the possible genetic compensation associated with this congenital disorder, however, it is difficult to establish a direct role for CBP in cognitive function in the adult brain. Although aspects of the clinical presentation in RSTS cases have been extensively studied, a spectrum of symptoms found in RSTS patients can be accessed only after birth, and, thus, prenatal genetic tests for this extremely rare genetic disorder are seldom considered. Even though there has been intensive research on the genetic and epigenetic function of the CREBBP gene in rodents, the etiology of this devastating congenital human disorder is largely unknown.

p300 (EP300) and CBP (CREBBP) are transcriptional coactivators with histone acetyltransferase activity. Various β-cell transcription factors can recruit p300/CBP, and thus the coactivators could be important for β-cell function and health in vivo. We hypothesized that p300/CBP contribute to the development and proper function of pancreatic islets. To test this, we bred and studied mice lacking p300/CBP in their islets. Mice lacking either p300 or CBP in islets developed glucose intolerance attributable to impaired insulin secretion, together with reduced α- and β-cell area and islet insulin content. These phenotypes were exacerbated in mice with only a single copy of p300 or CBP expressed in islets. Removing p300 in pancreatic endocrine progenitors impaired proliferation of neonatal α- and β-cells. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish α- and β-cell mass postnatally. Transcriptomic analyses revealed significant overlaps between p300/CBP-downregulated genes and genes downregulated in Hnf1α-null islets and Nkx2.2-null islets, among others. Furthermore, p300/CBP are important for the acetylation of H3K27 at loci downregulated in Hnf1α-null islets. We conclude that p300 and CBP are limiting cofactors for islet development, and hence for postnatal glucose homeostasis, with some functional redundancy.

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator–based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9–driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9–driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1753–62. ©2017 AACR.

Background/objectives Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease triggered by epigenetic alterations, including lysine acetylation at histone or non-histone proteins, affecting the stability or transcription of lipogenic genes. Although various natural dietary compounds have anti-lipogenic effects, their effects on the acetylation status and lipid metabolism in the liver have not been thoroughly investigated. Materials/methods Following oleic-palmitic acid (OPA)-induced lipid accumulation in HepG2 cells, the acetylation status of histone and non-histone proteins, HAT activity, and mRNA expression of representative lipogenic genes, including PPARγ, SREBP-1c, ACLY, and FASN, were evaluated. Furthermore, correlations between lipid accumulation and HAT activity for 22 representative natural food extracts (NExs) were evaluated. Results Non-histone protein acetylation increased following OPA treatment and the acetylation of histones H3K9, H4K8, and H4K16 was accelerated, accompanied by an increase in HAT activity. OPA-induced increases in the mRNA expression of lipogenic genes were down-regulated by C-646, a p300/CBP-specific inhibitor. Finally, we detected a positive correlation between HAT activity and lipid accumulation (Pearson's correlation coefficient = 0.604) using 22 NExs. Conclusions Our results suggest that NExs have novel applications as nutraceutical agents with HAT inhibitor activity for the prevention and treatment of NAFLD.

The inhibitory effect of histone acetyltransferase from methanol extract of Terminalia chebula Retz. fruit (TCME) was investigated in prostate cancer cell. TCME significantly inhibited histone acetyltransferase (HAT) activity by over 50% at 100 μg/mL concentration. TCME treatment repressed androgen receptor (AR) mediated tran-scription, mRNA level of AR target genes, prostate specific antigen (PSA) and NKX-3.1, as well as AR acetylation. Finally, the prostate cancer cell viability was dramatically reduced by TCME treatment at 0～100 μg/mL concentration. These results indicated that TCME, as a potent HAT inhibitor, could suppress prostate cancer cell growth by AR mediated transcription regulation. Key words: Terminalia chebula , prostate cancer, histone acetyltransferase, androgen receptor, acetylation Received 16 September 2013; Accepted 26 September 2013E-mail: creamut@suwon.ac.kr, Phone: 82-31-229-8194 서 론 전립선암은 최근 우리나라에서 가파른 발병률의 증가를 보이는 남성암으로 미국에서는 남성사망의 두 번째 원인으로 꼽히고 있다(1). 전립선암 발병에는 노화와 육식 위주 식습관에 따른 동물성지방 과다섭취 등의 식생활이 영향을 미치는 것으로 알려져 있다(2,3). 전립선암의 치료는 호르몬 요법이 전형적으로 사용되는데(4), 안드로젠은 남성호르몬으로 생식계의 발달, 유지, 성장에 필수적이며, 이 호르몬의 수용체인 안드로젠 수용체(androgen receptor, AR)는 전립선암의 발달과 깊은 연관이 있다(5). 최근 안드로젠 수용체(androgen receptor, AR)의 아세틸화, 인산화 등의 전사 후 변형(post transcrip-tional modification)이 전립선암세포의 성장과 관련이 있다고 밝혀지면서(6) 암과 연관된 히스톤 탈아세틸화 효소(histone deacetylase, HDAC) 및 히스톤 아세틸 전이효소(histone acetyltransferase, HAT) 등의 히스톤 변형 효소에 관한 연구가 진행되고 있다. p300, p/CAF, TIP60 등의 HAT 단백질은 AR을 직접적으로 아세틸화하고, 이는 co- activator binding을 강화하여(7), 결국 AR을 핵으로 translocation 함으로써 전립선암세포의 성장을 촉진시킨다고(4) 보고되었다. 그러므로 HAT 활성의 억제를 이용한 AR 아세틸화 조절은 새로운 전립선암 치료제 개발에서 중요한 기작이 될 수 있으며, 이러한 활성을 지닌 천연물의 발굴이 기존의 호르몬 치료와 병행할 수 있는 보완적인 치료제 개발의 기반이 될 수 있다.가자는 사군자과(Cambretaceae)에 속하는 Terminalia chebula Retz.의 성숙한 과실을 건조한 것으로 약용으로 사용되고 있다(8). 가자의 성분으로는 chebulic acid, chebu-lagic acid, gallic acid, tannin 등이 있다고 알려져 있으며(8), 항균(9), 항암(10), 항염 및 항산화 활성(11), 항당뇨 활성(12)이 있다고 알려져 있다. 본 연구에서는 가자 메탄올 추출물이 HAT 활성을 억제하고 AR의 아세틸화를 감소시킴으로써 전립선암세포의 성장을 저해하는 것을 밝히고, 전립선암 예방 치료의 소재 개발의 기초자료를 제공하고자 한다.

Gcn5 is a well-established histone acetyltransferase involved in chromatin modification by catalyzing the acetylation of specific lysine residues within the N-terminal tails of the core histones. To assess the role of chromatin remodeling in the transcriptional response of cellulolytic Trichoderma reesei to the changes of environmental conditions, we identified the T. reesei ortholog of Saccharomyces cerevisiae Gcn5 by sequence alignment and functional analysis. Heterologous expression of TrGcn5 in S. cerevisiae gcn5Δ strain restored the growth defect under nutrient limitation as well as stresses. In contrast, mutant TrGcn5 with site-directed changes of residues critical for Gcn5 histone acetyltransferase activity could not complement the growth defect. The T. reesei gcn5Δ mutant strain displayed a strongly decreased growth rate and dramatic morphological changes including misshapen hyphal cells and abolished conidiation. Moreover, the induced expression of cellulase genes was severely impaired in the gcn5Δ T. reesei with acetylation of K9 and K14 of histone H3 in the cellulase gene promoter dramatically affected in the absence of TrGcn5. The results indicate that TrGcn5 plays a critical role in filamentous growth, morphogenesis, and transcriptional activation of specific genes including cellulase encoding genes.

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm2, for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

The teratogenicity of the antiepileptic drug valproic acid (VPA) is well established and its inhibition of histone deacetylases (HDAC) is proposed as an initiating factor. Recently, VPA-mediated HDAC inhibition was demonstrated to involve transcriptional downregulation of histone acetyltransferases (HATs), which was proposed to compensate for the increased acetylation resulting from HDAC inhibition. Cbp and p300 are HATs required for embryonic development and deficiencies in either are associated with congenital malformations and embryolethality. The objective of the present study was to characterize Cbp/p300 following VPA exposure in P19 cells. Consistent with previous studies, exposure to 5mM VPA over 24h induced a moderate decrease in Cbp/p300 mRNA, which preceded a strong decrease in total cellular protein mediated by ubiquitin-proteasome degradation. Nuclear Cbp/p300 protein was also decreased following VPA exposure, although to a lesser extent. Total cellular and nuclear p300 HAT activity was reduced proportionately to p300 protein levels, however while total cellular HAT activity also decreased, nuclear HAT activity was unaffected. Using the Cbp/p300 HAT inhibitor C646, we demonstrated that HAT inhibition similarly affected many of the same endpoints as VPA, including increased reactive oxygen species and caspase-3 cleavage, the latter of which could be attenuated by pre-treatment with the antioxidant catalase. C646 exposure also decreased NF-κB/p65 protein, which was not due to reduced mRNA and was not attenuated with catalase pre-treatment. This study provides support for an adaptive HAT response following VPA exposure and suggests that reduced Cbp/p300 HAT activity could contribute to VPA-mediated alterations.

Following amino acid deprivation, the amino acid response (AAR) induces transcription from specific genes through a collection of signaling mechanisms, including the GCN2-eIF2-ATF4 pathway. The present report documents that the histone demethylase JMJD3 is an activating transcription factor 4 (ATF4)-dependent target gene. The JMJD3 gene contains two AAR-induced promoter activities and chromatin immunoprecipitation (ChIP) analysis showed that the AAR leads to enhanced ATF4 recruitment to the C/EBP-ATF response element (CARE) upstream of Promoter-1. AAR-induced histone modifications across the JMJD3 gene locus occur upon ATF4 binding. Jmjd3 transcription is not induced in Atf4-knock-out cells, but the AAR-dependent activation was rescued by inhibition of histone deacetylation with trichostatin A (TSA). The TSA rescue of AAR activation in the absence of Atf4 also occurred for the Atf3 and C/EBP homology protein (Chop) genes, but not for the asparagine synthetase gene. ChIP analysis of the Jmjd3, Atf3, and Chop genes in Atf4 knock-out cells documented that activation of the AAR in the presence of TSA led to specific changes in acetylation of histone H4. The results suggest that a primary function of ATF4 is to recruit histone acetyltransferase activity to a sub-set of AAR target genes. Thus, absolute binding of ATF4 to these particular genes is not required and no ATF4 interaction with the general transcription machinery is necessary. The data are consistent with the hypothesis that ATF4 functions as a pioneer factor to alter chromatin structure and thus, enhance transcription in a gene-specific manner.

Histone acetyltransferase enzymes (HATs) are important therapeutic targets, but there are few cell-based assays available for evaluating the pharmacodynamics of HAT inhibitors. Here we present the application of a FRET-based reporter, Histac, in live cell studies of p300/CBP HAT inhibition, by both genetic and pharmacologic disruption. shRNA knockdown of p300/CBP led to increased Histac FRET, suggesting a role for p300/CBP in the acetylation of the histone H4 tail present. Additionally, we describe a new p300/CBP HAT inhibitor, C107, and show that it can also increase cellular Histac FRET. Taken together, these studies provide a live cell strategy for identifying and evaluating p300/CBP inhibitors.

Alteration of the acetylation state of histone proteins contributes to transcriptional regulation and epigenetic inheritance. Dysregulation of these processes may lead to human diseases, especially cancer. One of the major chromatin modifications is histone acetylation and this review gives an overview of the role of histone acetyltransferases, their structural aspects, as well as of chemical modulators targeting their enzymatical activities. Inhibitors and activators of histone acetyltransferases are presented and their capability to influence histone and non-histone protein acetylation levels is discussed. Development of small molecules as epigenetic tools that alter histone acetyltransferase activity will be helpful to better understand the consequences of histone and generally protein acetylation and potentially offer novel therapeutic approaches for the treatment of cancer and other diseases.

Human β-defensin-2 (HBD-2) is an antimicrobial peptide produced by the epithelial cells that plays an important role in innate and adaptive immunity. Here we report that high mobility group protein N2 (HMGN2), a member of the high mobility group superfamily that affects chromatin function, modulates the expression of HBD-2 in A549 cells treated by lipopolysaccharide. Mechanistically, HMGN2 prolongs the retention time and enhances the accumulation of nuclear factor κB p65 in the nucleus, and promotes the acetylation of p65 through increasing histone acetyltransferase activity and enhancing p65-Ser536 phosphorylation. Additionally, chromatin immunoprecipitation reveals that HMGN2 and p65 synergistically promote their specific binding to HBD-2 promoter, thereby affecting the downstream transcription. Taken together, these results suggest that HMGN2 acts as a positive modulator of nuclear factor κB signalling to promote lipopolysaccharide-induced β-defensin expression.

Glucocorticoids influence organ functions through the glucocorticoid receptor, a protein acetylated and deacetylated by several histone acetyltransferases and deacetylases. We reported that the circadian rhythm-related transcription factor “Clock”, a key component of the biological CLOCK with inherent histone acetyltransferase activity, acetylates glucocorticoid receptor lysines within its hinge region—a “lysine cluster” containing a KXKK motif—and represses its transcriptional activity. This Clock-induced repression of the glucocorticoid receptor activity is inversely phased to the diurnally circulating glucocorticoids and may act as a local counter regulatory mechanism to the actions of these hormones. Importantly, uncoupling of the central CLOCK-regulated hypothalamic–pituitary–adrenal axis and peripheral CLOCK-mediated alterations of glucocorticoid action, such as chronic stress and frequent trans-time zone travel or night-shift work, may cause functional hypercortisolism and contribute to various pathologies. Thus, acetylation-mediated epigenetic regulation of the glucocorticoid receptor may be essential for the maintenance of proper time-integrated glucocorticoid action, significantly influencing human well-being and longevity.

Histone acetylation, which is regulated by histone acetyltransferases (HATs) and deacetylases, is an epigenetic mechanism that influences eukaryotic transcription. Significant changes in histone acetylation are associated with cancer; therefore, manipulating the acetylation status of key gene targets is likely crucial for effective cancer therapy. Grape seed extract (GSE) has a known protective effect against prostate cancer. Here, we showed that GSE significantly inhibited HAT activity by 30-80% in vitro (P .05). Furthermore, we demonstrated significant repression of androgen receptor (AR)-mediated transcription by GSE in prostate cancer cells by measuring luciferase activity using a pGL3-PSA construct bearing the AR element in the human prostate cancer cell line LNCaP (P .05). GSE treatment also decreased the mRNA level of the AR-regulated genes PSA and NKX 3.1. Finally, GSE inhibited growth of LNCaP cells. These results indicate that GSE potently inhibits HAT, leading to decreased AR-mediated transcription and cancer cell growth, and implicate GSE as a novel candidate for therapeutic activity against prostate cancer.

Background: SRT1720 and SRT2183 were described recently as activators of the NAD+-dependent deacetylase, SIRT1. These molecules enhanced metabolic function when administered to rodents at doses of 100-500 mg/kg/day, purportedly by activating SIRT1 enzymatic activity in various tissues; however, considerable controversy surrounds these claims. Results: We find that these molecules do not activate SIRT1 deacetylase activity when tested in a variety of enzymatic assay formats and conditions. The compounds effectively decrease acetylated p53 in cells treated with DNA damaging agents but do so in cells that lack SIRT1, calling into question their designation as direct activators of SIRT1. In contrast, we find that the compounds inhibit p300 histone acetyltransferase activity in vitro, suggesting a possible mechanism for their effects in vivo. Conclusion: Structural features of these molecules may account for false-positive activation using fluorescence-based assays.

MSCs possess the capacity of self-renewal and potential of differentiation into various kinds of specialized tissue cells including myocardiocytes. From self-renewing to oriented differentiation, chromatin is remodeled into heritable states that allow activation or maintain the repression of regulatory genes, which means specific genes in self-renewing switched off and specific genes in oriented differentiation activated (Bernstein et al. Cell 125:315-326, 2006). These epigenetic states are established and controlled largely by specific patterns of histone posttranslational modifications, in particular, histone acetylation (Li Nat Rev Genet 3:662-673, 2002). In cardiomyocyte differentiation of rat MSCs, we focused on Gcn5, which linked a known transcriptional coactivator with catalytic histone acetyltransferase activity (Brownell et al. Cell 84:843-851, 1996). To clarify participatory in vivo role of Gcn5, using an RNA interference (RNAi) strategy employing shRNA to specifically knockdown Gcn5 expression in MSCs, we found that HAT activity altered dynamically depended on the inhibition of Gcn5 during MSCs differentiation. Chromatin immunoprecipitation (ChIP) assay showed the increased binding of acetyl histone H3 to the early cardiomyocyte-specific genes GATA4 and NKx2.5 promoters in cardiomyocyte differentiation of MSCs by 5-azacytidine inducing, whereas the decreased binding with lower Gcn5 expression. Cell ultrastructure analysis revealed that MSCs induced by 5-azacytidine possess morphological characteristics of cardiomyocyte cells. The shape of MSCs transfected by Gcn5 RNAi was similar to normal MSCs, but the chromatin showed heavy electron-density and a hard-packed structure. This intermediate state of chromatin may be an inactive part of MSCs differentiation. These results demonstrate that Gcn5, possessing acetyltransferase activity, is involved in regulating chromatin configuration around GATA4 and NKx2.5 in cardiomyocyte differentiation of rat MSCs by changing the level of histone acetylation. HAT activity depending on Gcn5 is important in differentiation of MSCs into cardiomyocytes as a consequence of the remodeling of the chromatin configuration caused by modification of histone H3.

The bromodomain is an evolutionarily conserved motif found in many transcriptional activators including p300 which contains an intrinsic histone acetyltransferase activity and is a general coactivator for many transcription factors. One mode of bromodomain action is to serve as a binding module to recognize specific acetyl-lysine residue of histones during chromatin remodeling and transcriptional activation. The function of p300 is required for diverse sets of gene expression. However, it is not known whether the p300 bromodomain is involved in the expression of all or only subset of p300-dependent genes. In this study, we examined the impact of either a wild-type or a bromo-deficient p300 on the expression of several p300-dependant genes. The effects of histone acetylation on the expression of these genes were also assessed by targeting histone deacetylase activities with an inhibitor approach. We show that the impact of these inhibitors on the transcriptional activation of p300-dependent genes is impaired in cells containing the bromo-deficient p300, indicating that the interplay of p300 and histone acetylation in p300-dependent gene transcription requires the bromodomain. We also observed an increase in the expression of bromo-deficient p300 at the level of transcription possibly to compensate for the loss of p300 function. However, the high level of bromo-deficient p300 is not able to maintain the basal level of histone acetylation. Thus, the bromodomain is important for p300 to maintain the basal level of histone acetylation and to induce the transcriptional activation of p300-dependent genes. Nevertheless, the requirement of bromodomain and histone acetylation in p300-dependent gene transcription is determined by a gene specific manner.

The human T-cell leukemia virus, type-1 (HTLV-1)-encoded Tax protein is required for high-level transcription of the virus. Tax function is strictly dependent upon the phosphorylated form of the cellular transcription factor CREB (pCREB), and together they bind novel cAMP response elements located within the viral promoter. The DNA-bound Tax/pCREB complex recruits the cellular coactivators CBP/p300, which are essential for viral gene expression. The coactivators, via their histone acetyltransferase activity, function to promote changes in chromatin architecture that are permissive to transcriptional activation. Tax expression in vivo recruits p300 to the HTLV-1 promoter and correlates with depletion of nucleosomes from the integrated provirus. We recently developed a novel in vitro, chromatin-based experimental system that recapitulates the eviction of nucleosomes from the HTLV-1 promoter observed in vivo. These assays establish the essential function of Tax/pCREB recruitment of CBP/p300, and concomitant histone acetylation, in the nucleosome disassembly process. These observations are of particular significance, as Tax mediates disassembly of the full nucleosome octamer independent of transcriptional activity and ATP utilization. Instead, nucleosome eviction is absolutely dependent upon acetyl CoA and the histone chaperone Nap1. In this review, we will discuss HTLV-1, Tax transactivation, and our recent findings that uncover the critical role of Tax in promoting chromatin transitions that accompany activation of viral transcription. We will describe the phenomenon of acetylation-dependent promoter nucleosome disassembly and the emerging view that the formation of nucleosome-free promoter regions may represent a general prerequisite for transcriptional activation in eukaryotes.

SummaryIncreasing evidence indicates that epigenetic changes regulate cell genesis. Here, we ask about neural precursors, focusing on CREB binding protein (CBP), a histone acetyltransferase that, when haploinsufficient, causes Rubinstein-Taybi syndrome (RTS), a genetic disorder with cognitive dysfunction. We show that neonatal cbp+/− mice are behaviorally impaired, displaying perturbed vocalization behavior. cbp haploinsufficiency or genetic knockdown with siRNAs inhibited differentiation of embryonic cortical precursors into all three neural lineages, coincident with decreased CBP binding and histone acetylation at promoters of neuronal and glial genes. Inhibition of histone deacetylation rescued these deficits. Moreover, CBP phosphorylation by atypical protein kinase C ζ was necessary for histone acetylation at neural gene promoters and appropriate differentiation. These data support a model in which environmental cues regulate CBP activity and histone acetylation to control neural precursor competency to differentiate, and indicate that cbp haploinsufficiency disrupts this mechanism, thereby likely causing cognitive dysfunction in RTS.

Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.

A simple radiometric assay for histone acetyltransferase (HAT) activity employing a PhosphorImager is described. In the proposed procedure, following incubation of [1-(14)C]acetyl coenzyme A (CoA), histones, and HAT enzyme, radiolabeled histones are fixed on GF/F glass microfiber filter while the excess of acetyl CoA is washed out. Afterward, the filter is exposed to a phosphor-screen and the resulting spot signals are quantified with a PhosphorImager. Given the small volumes required, the new assay reduces reagent consumption and contaminated waste. Moreover, the assay can be performed with a large number of samples simultaneously, is applicable on different protein substrates, and is adaptable to the analysis of other protein modifications.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex of Saccharomyces cerevisiae contains more than 20 components that acetylate and deubiquitylate nucleosomal histones. Its acetyltransferase, Gcn5, preferentially acetylates histones H3 and H2B and is regulated through interactions with Ada2 and Ngg1/Ada3. Sequence alignments of Ada2 homologs indicate a conserved approximately 120-amino-acid-residue central region. To examine the function of this region, we constructed ada2 alleles with mutations of clustered conserved residues. One of these alleles, ada2-RLR (R211S, L212A, and R215A), resulted in an approximately threefold reduction in transcriptional activation of the PHO5 gene and growth changes that parallel deletion of ada2. Microarray analyses further revealed that ada2-RLR alters expression of a subset of those genes affected by deletion of ada2. Indicative of Ada2-RLR affecting Gcn5 function, Ada2-RLR resulted in a decrease in Gcn5-mediated histone acetylation in vitro to a level approximately 40% that with wild-type Ada2. In addition, in vivo acetylation of K16 of histone H2B was almost totally eliminated at Ada2-regulated promoters in the ada2-RLR strain, while acetylation of K9 and K18 of histone H3 was reduced to approximately 40% of wild-type levels. We also show that the central region of Ada2 interacts with phospholipids. Since phosphatidylserine binding paralleled Ada2 function, we suggest that lipid binding may play a role in the function or regulation of the SAGA complex.

Histone modifications affect chromatin dynamics on several levels by serving as binding sites for regulatory proteins. In many cell types, including embryonic stem cells (ESCs), a subset of genes is marked with histone modifications thought to be both activating and repressing: H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3), respectively. As a result, genes bearing this “bivalent” mark are transcribed at low levels, but are primed for activation, should the cell receive the appropriate cues during differentiation. Recently, we found that the Tip60-p400 acetyltransferase and histone exchange complex is necessary to maintain normal self-renewal in mouse ESCs. While Tip60-p400 has histone acetyltransferase activity, which is generally associated with transcriptional activation, it acts predominantly as a repressor of genes expressed during differentiation. Surprisingly, in ESCs Tip60-p400 localizes to the promoters of genes marked by H3K4me3, which include both highly expressed genes and “bivalent” genes expressed at low levels. Tip60-p400 acetylates histones at these targets, including the promoters for developmental regulators it helps to silence in ESCs. This suggests that the effect of chromatin modifications on transcription is not always simply positive or negative. Rather, we propose that the impact of specific modifications at each promoter is determined by the chromatin context in which they are found.