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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.

Lysophosphatidic acid receptor 6 (LPAR6) is a G protein–coupled receptor that plays critical roles in cellular morphology and hair growth. Although LPAR6 overexpression is also critical for cancer cell proliferation, its role in liver cancer tumorigenesis and the underlying mechanism are poorly understood. Here, using liver cancer and matched paracancerous tissues, as well as functional assays including cell proliferation, quantitative real-time PCR, RNA-Seq, and ChIP assays, we report that LPAR6 expression is controlled by a mechanism whereby hepatocyte growth factor (HGF) suppresses liver cancer growth. We show that high LPAR6 expression promotes cell proliferation in liver cancer. More importantly, we find that LPAR6 is transcriptionally down-regulated by HGF treatment and that its transcriptional suppression depends on nuclear receptor coactivator 3 (NCOA3). We note that enrichment of NCOA3, which has histone acetyltransferase activity, is associated with histone 3 Lys-27 acetylation (H3K27ac) at the LPAR6 locus in response to HGF treatment, indicating that NCOA3 transcriptionally regulates LPAR6 through the HGF signaling cascade. Moreover, depletion of either LPAR6 or NCOA3 significantly inhibited tumor cell growth in vitro and in vivo (in mouse tumor xenograft assays), similar to the effect of the HGF treatment. Collectively, our findings indicate an epigenetic link between LPAR6 and HGF signaling in liver cancer cells, and suggest that LPAR6 can serve as a biomarker and new strategy for therapeutic interventions for managing liver cancer.

Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb-group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here, we show that the p300/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been reported to exert protective effects against myocardial, hepatic, and gastric ischemia-reperfusion injury (IRI), but whether it can protect against renal IRI remains unknown. Here, a lethal renal IRI model was established with a 100% mortality rate in untreated mice. Treatment with liraglutide involving a regimen of multiple doses resulted in 100% survival, remarkable preservation of renal function, a significant reduction in pathological damage, and blunted upregulation of TNF-α, IL-1β, IL-6, MCP-1, TLR-2, TLR-4, and RAGE mRNA. We found that liraglutide treatment dramatically inhibited ischemia-induced nucleocytoplasmic translocation and release of HMGB1. This inhibition was associated with a marked decrease (~ 60%) in nuclear histone acetyltransferase activity. In addition, the protective effects of liraglutide on renal IRI were largely abolished by the administration of exogenous HMGB1. When the GLP-1R antagonist exendin (9-39) was given to mice before each liraglutide administration, or GLP-1R-/- mice were used for the renal IRI experiments, the protective effect of liraglutide on renal IRI was partially reversed. Moreover, liraglutide pretreatment significantly inhibited HMGB1 nucleocytoplasmic translocation during hypoxic culture of HK-2 cells in vitro, but the addition of exendin (9-39) significantly eliminated this inhibition. We demonstrate here that liraglutide can exert a strong protective effect on lethal renal IRI in mice. This protection appears to be related to the inhibition of HMGB1 nuclear-cytoplasmic translocation and release and partially depends on GLP-1R. Thus, liraglutide may be therapeutically useful for the clinical prevention and treatment of organ IRI.

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.

3,3′,4′,5,7-Pentahydroxyflavone-3-rhamnoglucoside (rutin) is a flavonoid with a wide range of pharmacological activities. Dietary rutin is hardly absorbed because the microflora in the large intestine metabolize rutin into a variety of compounds including quercetin and phenol derivatives such as 3,4-dihydroxyphenolacetic acid (DHPAA), 3,4-dihydroxytoluene (DHT), 3,4-hydroxyphenylacetic acid (HPAA) and homovanillic acid (HVA). We examined the potential of rutin and its metabolites as novel histone acetyltransferase (HAT) inhibitors. DHPAA, HPAA and DHT at the concentration of 25 μM significantly inhibited in vitro HAT activity with DHT having the strongest inhibitory activity. Furthermore, DHT was shown to be a highly efficient inhibitor of p300 HAT activity, which corresponded with its high degree of inhibition on intracellular lipid accumulation in HepG2 cells. Docking simulation revealed that DHT was bound to the p300 catalytic pocket, bromodomain. Drug affinity responsive target stability (DARTS) analysis further supported the possibility of direct binding between DHT and p300. In HepG2 cells, DHT concentration-dependently abrogated p300-histone binding and induced hypoacetylation of histone subunits H3K9, H3K36, H4K8 and H4K16, eventually leading to the downregulation of lipogenesis-related genes and attenuating lipid accumulation. In ob/ob mice, administration of DHT (10, 20 mg/kg, iv, every other day for 6 weeks) dose-dependently improved the NAFLD pathogenic features including body weight, liver mass, fat mass, lipid accumulation in the liver, and biochemical blood parameters, accompanied by the decreased mRNA expression of lipogenic genes in the liver. Our results demonstrate that DHT, a novel p300 histone acetyltransferase inhibitor, may be a potential preventive or therapeutic agent for NAFLD.

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.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial lung disease with a poor prognosis similar to that of malignancy. The causes of IPF are not clearly known, and there is no effective therapy to date. In this study, the natural compound plumbagin, which was isolated from Plumbago rosea root extract, was screened for p300 inhibitory activity. Plumbagin specifically inhibited the activity of p300 toward histone acetyltransferases. Plumbagin treatment significantly suppressed transforming growth factor-β-induced profibrotic target-gene expression and proliferation of fibroblast cell lines. Moreover, plumbagin significantly inhibited bleomycin-induced pulmonary fibrosis in mice. Taken together, these data demonstrate the inhibitory effects of plumbagin on lung fibrosis and its promise as a therapeutic agent for IPF.

Mastitis is a major inflammatory response of the mammary gland due to various pathogenic invasions and is a serious disease that affects the production yield and health status of cows. Astaxanthin (AST), a xanthophyll carotenoid, is a secondary metabolite synthesized by microalgae and yeasts that has been reported to suppress various inflammatory responses. However, the protective effect of AST on lipopolysaccharide (LPS)-induced mammary epithelial cells has not yet been reported. The present study results indicated that AST treatment markedly attenuated the oxidative stress markers and nitric oxide (NO) while improving the anti-oxidant enzymes in LPS exposed cells. On the other hand, LPS-exposed cells showed nuclear translocation of nuclear factor-κB (NF-κB) with the activation of inflammatory cytokines such as monocyte chemoattractant protein-1, tumor necrosis factor-α, interferon-γ, and interleukin-6 (IL-6). In addition, mRNA expression analysis revealed that the histone deacetylase (HDAC) -1, -2, -3, -6, -7 and pentraxin 3 (PTX3) expressions were increased in the LPS group. Furthermore, the activity of HDAC was increased to 2-fold with a significant reduction in the histone acetyltransferase activity in cells exposed to LPS. However, AST was able to inhibit the nuclear translocation of NF-κB with attenuated HDAC activity. Intriguingly, HDAC inhibition studies demonstrated that the cytokines such as IL-4, IL-8, granulocyte-mcrophage colony stimulating factor, C-reactive protein, IL-17A, and IL-22 were significantly suppressed which were upregulated in LPS treatment; while AST was found acting by improving the anti-inflammatory cytokine IL-10, and thioredoxin reductase levels. Collectively, these findings provide novel insights into the role of HDACs in regulating cellular processes involved in the pathogenesis of LPS-induced mastitis as well as the potential use of AST as a therapeutic in treatment for controlling disease progression.

Chronic stress leads to post-traumatic stress disorder (PTSD) and metabolic disorders including fatty liver. We hypothesized that stress-induced molecular mechanisms alter energy metabolism, thereby promoting hepatic lipid accumulation even after a stress-free recovery period. In this context, we investigated fibroblast growth factor-21 (FGF21) as protective for energy and glucose homeostasis. FGF21 knockout mice (B6.129S6(SJL)-Fgf21tm1.2Djm; FGF21KO) and control mice (C57BL6; WT) were subjected to chronic variable stress. Mice were examined directly after acute intervention (Cvs) and long-term after 3 months of recovery (3mCvs). In WT, Cvs reduced insulin sensitivity and hepatic lipid accumulation, whilst fatty acid uptake increased. FGF21KO mice responded to Cvs with improved glucose tolerance, insulin resistance but liver triglycerides and plasma lipids were unaltered. Hepatic gene expression was specifically altered by genotype and stress e.g. by PPARa and SREBP-1 regulated genes. The stress-induced alteration of hepatic metabolism persisted after stress recovery. In hepatocytes at 3mCvs, differential gene regulation and secreted proteins indicated a genotype specific progression of liver dysfunction. Overall, at 3mCvs FGF21 was involved in maintaining mitochondrial activity, attenuating de novo lipogenesis, increased fatty acid uptake and histone acetyltransferase activity. Glucocorticoid release and binding to the FGF21 promoter may contribute to prolonged FGF21 release and protection against hepatic lipid accumulation. In conclusion, we showed that stress favors fatty liver disease and FGF21 protected against hepatic lipid accumulation after previous chronic stress loading by i) restored physiological function, ii) modulated gene expression via DNA-modifying enzymes, and iii) maintained energy metabolism.

Several studies have demonstrated that the intrinsic ability of neurons to regenerate their axons can be stimulated by maneuvers that favor the open state of chromatin, such as inhibiting histone deacetylase activity or increasing histone acetyltransferase activity. Taken together, these experiments suggest that axon regenerative ability can be increased by promoting chromatin accessibility. In this article, we assess the direct evidence in the literature for this hypothesis and re-examine other axon regeneration-promoting manipulations to see if they provide additional support. We find that several interventions known to enhance intrinsic axonal growth capability also increase chromatin accessibility. Although the support for this correlation is strong in the literature, we conclude with a word of caution about therapeutics attempting to exploit this relationship.

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.

Lysine acetylations are post-translational modifications of cellular proteins, that are crucial in the regulation of many cellular processes. Lysine acetylations on histone proteins are part of the epigenetic code regulating gene expression and are installed by histone acetyltransferases. Observations that inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, are characterized by increased histone acetyltransferase activity indicate that development of small molecule inhibitors for these enzymes might be a valuable approach towards new therapies for these diseases. The 6-alkylsalicylate MG149 is a candidate to explore this hypothesis because it has been demonstrated to inhibit the MYST type histone acetyltransferases. In this study, we determined the K i value for inhibition of the MYST type histone acetyltransferase KAT8 by MG149 to be 39 ± 7.7 μM. Upon investigating whether the inhibition of histone acetyltransferases by MG149 correlates with inhibition of histone acetylation in murine precision-cut lung slices, inhibition of acetylation was observed using an LC-MS/MS based assay on histone H4 res 4–17, which contains the target lysine of KAT8. Following up on this, upon treatment with MG149, reduced pro-inflammatory gene expression was observed in lipopolysaccharide and interferon gamma stimulated murine precision-cut lung slices. Based on this, we propose that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases.

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.

K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

Background The etiology of bipolar disorder (BD) is multifactorial, involving both environmental and genetic factors. Current pharmacological treatment is associated with several side effects, which are the main reason patients discontinue treatment. Epigenetic alterations have been studied for their role in the pathophysiology of BD, as they bridge the gap between gene and environment. Objective Evaluate the effects of histone deacetylase inhibitors on behavior and epigenetic enzymes activity in a rat model of mania induced by ouabain. Methods Adult male rats were subjected to a single intracerebroventricular injection of ouabain (10−3 M) followed by 7 days of valproate (200 mg/kg) or sodium butyrate (600 mg/kg) administration. Locomotor and exploratory activities were evaluated in the open-field test. Histone deacetylase, DNA methyltransferase, and histone acetyltransferase activity were assessed in the frontal cortex, hippocampus, and striatum. Results Ouabain induced hyperactivity in rats, which was reversed by valproate and sodium butyrate treatment. Ouabain did not alter the activity of any of the enzymes evaluated. However, valproate and sodium butyrate decreased the activity of histone deacetylase and DNA methyltransferase. Moreover, there was a positive correlation between these two enzymes. Conclusion These results suggest that targeting epigenetic mechanisms may play an important role in mania-like behavior management.

Alcohol exposure during development produces physical and mental abnormalities in the foetus that result in long-term molecular adjustments in the brain, which could underlie the neurobehavioural deficits observed in individuals suffering from foetal alcohol spectrum disorders. In this study, we assessed the effects of curcumin on cognitive impairments caused by prenatal and lactational alcohol exposure (PLAE). Furthermore, we examined whether curcumin could counteract the molecular alterations that may underlie these behavioural impairments. We focused on inflammatory and epigenetic mechanisms by analysing the expression of pro-inflammatory mediators, such as IL-6, TNF-α, and NF-κB, in the hippocampus and prefrontal cortex, as well as microglia and astrocyte activation in the dentate gyrus. We also assessed the activity of histone acetyltransferase in these brain areas. To model binge alcohol drinking, we exposed pregnant C57BL/6 mice to a 20% v/v alcohol solution during gestation and lactation, with limited access periods. We treated male offspring with curcumin during postnatal days (PD28-35) and then evaluated their behaviour in adulthood (PD60). Our results showed that curcumin treatment during the peri-adolescence period improved the anxiety and memory deficits observed in PLAE mice. At the molecular level, we found enhanced histone acetyltransferase activity in mice subjected to PLAE that curcumin treatment could not reverse to baseline levels. These mice also showed increased expression of pro-inflammatory mediators, which could be rescued by curcumin treatment. They also displayed astrogliosis and microglia activation. Our study provides further evidence to support the use of curcumin as a therapeutic agent for counteracting behavioural and molecular alterations induced by PLAE.

Even though Entamoeba histolytica (Eh)-induced host pro-inflammatory responses play a critical role in disease, we know very little about the host factors that regulate this response. Direct contact between host cell and Eh signify the highest level of danger, and to eliminate this threat, the host immune system elicits an augmented immune response. To understand the mechanisms of this response, we investigated the induction and release of the endogenous alarmin molecule high-mobility group box 1 (HMGB1) that act as a pro-inflammatory cytokine and chemoattractant during Eh infection. Eh in contact with macrophage induced a dose- and time-dependent secretion of HMGB1 in the absence of cell death. Secretion of HMGB1 was facilitated by Eh surface Gal-lectin-activated phosphoinositide 3-kinase and nuclear factor-κB signaling and up-regulation of histone acetyltransferase activity to trigger acetylated HMGB1 translocation from the nucleus. Unlike lipopolysaccharide, Eh-induced HMGB1 release was independent of caspase-1-mediated inflammasome and gasdermin D pores. In vivo, Eh inoculation in specific pathogen-free but not germ-free mice was associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and keratinocyte-derived chemokine, which was suppressed with HMGB1 neutralization. This study reveals that Eh-induced active secretion of the HMGB1 plays a key role in shaping the pro-inflammatory landscape critical in innate host defense against amebiasis.

Cellular senescence contributes to aging and age-related disorders. High glucose (HG) induces mesenchymal stromal/stem cell (MSC) senescence, which hampers cell expansion and impairs MSC function. Intracellular HG triggers metabolic shift from aerobic glycolysis to oxidative phosphorylation, resulting in reactive oxygen species (ROS) overproduction. It causes mitochondrial dysfunction and morphological changes. Tryptophan metabolites such as 5-methoxytryptophan (5-MTP) and melatonin attenuate HG-induced MSC senescence by protecting mitochondrial integrity and function and reducing ROS generation. They upregulate the expression of antioxidant enzymes. Both metabolites inhibit stress-induced MSC senescence by blocking p38 MAPK signaling pathway, NF-κB, and p300 histone acetyltransferase activity. Furthermore, melatonin upregulates SIRT-1, which reduces NF-κB activity by de-acetylation of NF-κB subunits. Melatonin and 5-MTP are a new class of metabolites protecting MSCs against replicative and stress-induced cellular senescence. They provide new strategies to improve the efficiency of MSC-based therapy for diverse human diseases.

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

p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic β-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate.

// 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.

Neuroinflammation after stroke significantly contributes to neuronal cell death. Bromodomain and Extra Terminal Domain (BET) proteins are essential to inflammatory gene transcription. BET proteins (BRD2, BRD3, BRD4, and BRDT) have varied effects including chromatin remodeling, histone acetyltransferase activity, and as scaffolds to recruit transcription factors; they couple chromatin remodeling with transcription. BRD2/4 are of particularly interest to stroke-induced neuroinflammation that contributes to delayed cell death as they are required for NF-κB-dependent gene transcription. We hypothesized that targeting BET proteins for degradation with dBET1, a proteolysis targeting chimera (PROTAC) that combines the highly selective BET inhibitor JQ1 and a ligand for cereblon E3 ubiquitin ligase, will reduce brain injury in ischemic stroke. Male aged mice (18–20 months old) were subjected to permanent occlusion of the middle cerebral artery and received either vehicle or dBET1 (10 mg/kg; i.p.) at various times after stroke. Neurobehavioral tests were performed before (baseline) and at 24 and 48 h after stroke induction. Infarct volume was quantified at 48 h. Data showed that BET degradation significantly reduced infarct volume in permanent focal cerebral ischemia in aged mice, and this was associated with reduced brain levels of pro-inflammatory mediators including TNF-α, CXCL1, CXCL10, CCL2, and matrix metalloproteinase-9. Treatment with dBET1 significantly reduced blood-brain barrier damage and infiltration of neutrophils into the ischemic brain. Importantly, treatment with the BET degrader dBET1 resulted in a significant improvement in stroke-induced neurological deficits. Collectively, these data indicate that BET proteins are a novel target for neuroprotection in ischemic stroke.

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.

Bisphenol A (BPA) is an endocrine disruptor whose ubiquitous presence in the environment has been related with impairment of male reproduction. BPA can cause both transcriptomic and epigenetic changes during spermatogenesis. To evaluate the potential effects of male exposure to BPA, adult zebrafish males were exposed during spermatogenesis to doses of 100 and 2000 μg/L, which were reported in contaminated water bodies and higher than those allowed for human consumption. Fertilization capacity and survival at hatching were analysed after mating with untreated females. Spermatogenic progress was analysed through a morphometrical study of testes and apoptosis was evaluated by TUNEL assay. Testicular gene expression was evaluated by RT-qPCR and epigenetics by using ELISA and immunocytochemistry. In vitro studies were performed to investigate the role of Gper. Chromatin fragmentation and the presence of transcripts were also evaluated in ejaculated sperm. Results on testes from males treated with the highest dose showed a significant decrease in spermatocytes, an increase in apoptosis, a downregulation of ccnb1 and sycp3, all of which point to an alteration of spermatogenesis and to meiotic arrest and an upregulation of gper1 and esrrga receptors. Additionally, BPA at 2000 μg/L caused missregulation of epigenetic remodelling enzymes transcripts in testes and promoted DNA hypermethylation and H3K27me3 demethylation. BPA also triggered an increase in histone acetyltransferase activity, which led to hyperacetylation of histones (H3K9ac, H3K14ac, H4K12ac). In vitro reversion of histone acetylation changes using a specific GPER antagonist, G-36, suggested this receptor as mediator of histone hyperacetylation. Males treated with the lower dose only showed an increase in some histone acetylation marks (H3K14ac, H4K12ac) but their progeny displayed very limited survival at hatching, revealing the deleterious effects of unbalanced paternal epigenetic information. Furthermore, the highest dose of BPA led to chromatin fragmentation, promoting direct reproductive effects, which are incompatible with embryo development.

The evolutionarily conserved Elongator Complex associates with RNA polymerase II for transcriptional elongation. Elp3 is the catalytic subunit, contains histone acetyltransferase activity, and is associated with neurodegeneration in humans. Elp1 is a scaffolding subunit and when mutated causes familial dysautonomia. Here, we show that elp3 and elp1 are required for aversive long-term olfactory memory in Drosophila. RNAi knockdown of elp3 in adult mushroom bodies impairs long-term memory (LTM) without affecting earlier forms of memory. RNAi knockdown with coexpression of elp3 cDNA reverses the impairment. Similarly, RNAi knockdown of elp1 impairs LTM and coexpression of elp1 cDNA reverses this phenotype. The LTM deficit in elp3 and elp1 knockdown flies is accompanied by the abolishment of a LTM trace, which is registered as increased calcium influx in response to the CS+ odor in the α-branch of mushroom body neurons. Coexpression of elp1 or elp3 cDNA rescues the memory trace in parallel with LTM. These data show that the Elongator complex is required in adult mushroom body neurons for long-term behavioral memory and the associated long-term memory trace.

Tobacco smoke is a source of many xenobiotics and free radicals. Reactive oxygen species can affect the body both directly and indirectly, through the activation of both signalling pathways and transcription factors (NF-κB and AP-1). One of the most important signalling cascades which can affect the oxidants in smoke are mitogen-activated protein kinases (MAPK). The mechanism of MAPK pathways activation by reactive oxygen species depends on the stimulation of specific tyrosine kinases and protein tyrosine phosphatases inactivation. An activated MAP protein can initiate AP-1 signalling and interact with many other transcription factors. The components of tobacco smoke with oxidation-reduction properties can have an effect on NF-κB signalling. Binding of NF-κB and AP-1 with DNA is a complicated process, in which coactivators exhibiting internal histone acetyltransferase activity are involved. The balance between histone deacetylases and acetylases is important for the regulation of inflammatory response in the lungs. Tobacco smoke causes increased acetylase activity and decreased deacetylase activity in epithelial lung cells. The result is an increase in the activation of NF-κB and AP-1. Oxygen free radicals from tobacco smoke can change the redox status of cells, which can in turn induce the activation of transcription factors, chromatin remodelling and intensified genes transcription for inflammatory mediators.

Aim: To assess the function of Ngg1 in Candida albicans and reveal the role of NGG1 in the morphological conversion and virulence of C. albicans. Materials & methods: C. albicans NGG1 gene was deleted in the wild-type strain SC5314 and the function of Ngg1 was assessed by western blot analysis. The phenotypes and the virulence of the ngg1 mutants were examined. Microarray analysis was performed to explore the mechanism. Results: The ngg1 mutants attenuated acetylated histone H3, obviously reduced filamentous growth and showed significantly diminished pathogenicity in all the infection models. Conclusion: This study suggested the histone acetyltransferase activity of C. albicans Ngg1 and revealed the important role of NGG1 in morphological conversion and virulence of C. albicans. [Formula: see text]

In a high percentage (≥85%) of both sporadic and familial adenomatous polyposis forms of colorectal cancer (CRC), the inactivation of the APC tumor suppressor gene initiates tumor formation and modulates the Wnt/β-Catenin transduction pathways involved in the control of cell proliferation, adhesion and metastasis. Increasing evidence showed that the endocannabinoids control tumor growth and progression, both in vitro and in vivo. We evaluated the effect of Rimonabant, a Cannabinoid Receptor 1 (CB1) inverse agonist, on the Wnt/β-Catenin pathway in HCT116 and SW48 cell lines carrying the genetic profile of metastatic CRC poorly responsive to chemotherapies. In these models, Rimonabant inhibited the Wnt/β-Catenin canonical pathway and increased β-Catenin phosphorylation; in HCT116 cells, but not in SW48, the compound also triggered the Wnt/β-Catenin non canonical pathway activation through induction of Wnt5A and activation of CaMKII. The Rimonabant-induced downregulation of Wnt/β-Catenin target genes was partially ascribable to a direct inhibition of p300/KAT3B histone acetyltransferase, a coactivator of β-Catenin dependent gene regulation. Finally, in HCT116 xenografts, Rimonabant significantly reduced tumor growth and destabilized the nuclear localization of β-Catenin. Obtained data heavily supported the rationale for the use of cannabinoids in combined therapies for metastatic CRC harbouring activating mutations of β-Catenin.

Preterm birth is a major contributor to early and delayed physical and cognitive impairment. Epidemiological and experimental data indicate that maternal infections are a significant and preventable cause of preterm birth. Recently, melatonin has been suggested to exert neuroprotective effects in several models of brain injury. Here, we sought to investigate whether the administration of melatonin is able to prevent lipopolysaccharide (LPS)-induced fetal brain damage in a model of LPS-induced preterm labor. For this purpose, 15-day pregnant BALB/c mice received intraperitoneally 2 doses of LPS or vehicle: the first one at 10:00 hours (0.26 mg/kg) and the second at 13:00 hours (0.52 mg/kg). On day 14 of pregnancy, a group of mice was subcutaneously implanted with a pellet of 25 mg melatonin. This experimental protocol resulted in 100% of preterm birth and pup death in the LPS group and a 50% of term birth and pup survival in the melatonin + LPS group. In the absence of melatonin, fetuses from LPS-treated mothers showed histological signs of brain damage, microglial/macrophage activation, and higher levels of IL-1β, inducible nitric oxide synthase (NOS), and neuronal NOS mRNAs as well as increased histone acetyltransferase activity and histone H3 hyperacetylation. In contrast, antenatal administration of melatonin prevented LPS-induced fetal brain damage. Moreover, when behavioral traits were analyzed in the offspring from control, melatonin, and melatonin + LPS, no significant differences were found, suggesting that melatonin prevented LPS-induced long-term neurodevelopmental impairments. Collectively, our results suggest that melatonin could be a new therapeutic tool to prevent fetal brain damage and its long-term consequences induced by maternal inflammation. Fil: Domínguez Rubio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Correa, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Aisemberg, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Dorfman, Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Bariani, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Rosenstein, Ruth Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Zorrilla Zubilete, María Aurelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina

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.

Since domestication, the genome landscape of cattle has been changing due to natural and artificial selection forces resulting in several general and specialized cattle breeds of the world. Identifying genomic regions affected due to these forces in livestock gives an insight into the history of selection for economically important traits and genetic adaptation to specific environments of the populations under consideration. This study explores the genes/genomic regions under selection in relation to the phenotypes of Holstein, Hanwoo, and N’Dama cattle breeds using Tajima’s D, XP-CLR, and XP-EHH population statistical methods. The whole genomes of 10 Holstein (South Korea), 11 Hanwoo (South Korea), and 10 N’Dama (West Africa—Guinea) cattle breeds re-sequenced to ~11x coverage and retained 37 million SNPs were used for the study. Selection signature analysis revealed 441, 512, and 461 genes under selection from Holstein, Hanwoo, and N’Dama cattle breeds, respectively. Among all these, seven genes including ARFGAP3, SNORA70, and other RNA genes were common between the breeds. From each of the gene lists, significant functional annotation cluster terms including milk protein and thyroid hormone signaling pathway (Holstein), histone acetyltransferase activity (Hanwoo), and renin secretion (N’Dama) were enriched. Genes that are related to the phenotypes of the respective breeds were also identified. Moreover, significant breed-specific missense variants were identified in CSN3, PAPPA2 (Holstein), C1orf116 (Hanwoo), and COMMD1 (N’Dama) genes. The genes identified from this study provide an insight into the biological mechanisms and pathways that are important in cattle breeds selected for different traits of economic significance.

The airway mucosa expresses protective interferon (IFN) and inflammatory cytokines in response to respiratory syncytial virus (RSV) infection. In this study, we examine the role of bromodomain containing 4 (BRD4) in mediating this innate immune response in human small airway epithelial cells. We observe that RSV induces BRD4 to complex with NF-κB/RelA. BRD4 is functionally required for expression of the NF-κB-dependent inflammatory gene regulatory network (GRN), including the IFN response factor 1 (IRF1) and IRF7, which mediate a cross talk pathway for RIG-I upregulation. Mechanistically, BRD4 is required for cyclin-dependent kinase 9 (CDK9) recruitment and phospho-Ser 2 carboxy-terminal domain (CTD) RNA polymerase (Pol) II formation on the promoters of IRF1 , IRF7 , and RIG-I , producing their enhanced expression by transcriptional elongation. We also find that BRD4 independently regulates CDK9/phospho-Ser 2 CTD RNA Pol II recruitment to the IRF3-dependent IFN-stimulated genes (ISGs). In vivo , poly(I·C)-induced neutrophilia and mucosal chemokine production are blocked by a small-molecule BRD4 bromodomain inhibitor. Similarly, BRD4 inhibition reduces RSV-induced neutrophilia, mucosal CXC chemokine expression, activation of the IRF7-RIG-I autoamplification loop, mucosal IFN expression, and airway obstruction. RSV infection activates BRD4 acetyltransferase activity on histone H3 Lys (K) 122, demonstrating that RSV infection activates BRD4 in vivo . These data validate BRD4 as a major effector of RSV-induced inflammation and disease. BRD4 is required for coupling NF-κB to expression of inflammatory genes and the IRF-RIG-I autoamplification pathway and independently facilitates antiviral ISG expression. BRD4 inhibition may be a strategy to reduce exuberant virus-induced mucosal airway inflammation. IMPORTANCE In the United States, 2.1 million children annually require medical attention for RSV infections. A first line of defense is the expression of the innate gene network by infected epithelial cells. Expression of the innate response requires the recruitment of transcriptional elongation factors to rapidly induce innate response genes through an unknown mechanism. We discovered that RSV infection induces a complex of bromodomain containing 4 (BRD4) with NF-κB and cyclin-dependent kinase 9 (CDK9). BRD4 is required for stable CDK9 binding, phospho-Ser 2 RNA Pol II formation, and histone acetyltransferase activity. Inhibition of BRD4 blocks Toll-like receptor 3 (TLR3)-dependent neutrophilia and RSV-induced inflammation, demonstrating its importance in the mucosal innate response in vivo . Our study shows that BRD4 plays a central role in inflammation and activation of the IRF7-RIG-I amplification loop vital for mucosal interferon expression. BRD4 inhibition may be a strategy for modulating exuberant mucosal airway inflammation.

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.

Sleep fragmentation (SF) is a common condition among pregnant women, particularly during late gestation. Gestational perturbations promote the emergence of adiposity and metabolic disease risk in offspring, most likely through epigenetic modifications. Adiponectin (AdipoQ) expression inversely correlates with obesity and insulin resistance. The effects of SF during late gestation on metabolic function and AdipoQ expression in visceral white adipose tissue (VWAT) of offspring mice are unknown. Male offspring mice were assessed at 24 weeks after dams were exposed to SF or control sleep during late gestation. Increased food intake, body weight, VWAT mass, and insulin resistance, with reductions in AdipoQ expression in VWAT, emerged in SF offspring. Increased DNMT3a and -b and global DNA methylation and reduced histone acetyltransferase activity and TET1, -2, and -3 expression were detected in VWAT of SF offspring. Reductions in 5-hydroxymethylcytosine and H3K4m3 and an increase in DNA 5-methylcytosine and H3K9m2 in the promoter and enhancer regions of AdipoQ emerged in adipocytes from VWAT and correlated with AdipoQ expression. SF during late gestation induces epigenetic modifications in AdipoQ in male offspring mouse VWAT adipocytes along with a metabolic syndrome–like phenotype. Thus, altered gestational environments elicited by SF impose the emergence of adverse, long-lasting metabolic consequences in the next generation.

Background: The mechanism driving hepatic gene expression of Foxo1 in the fasted state remains unclear. Results: Activation of cAMP-PKA pathway induces Foxo1 gene expression through CREB and co-activator P300. Conclusion: P300 mediates Foxo1 gene expression by binding to Foxo1 proximal promoter. Significance: Induction of the Foxo1 gene by cAMP-PKA via P300 fully activates the gluconeogenic program during fasting to maintain euglycemia., FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16 h of fasting. Furthermore, dibutyrl cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to tandem cAMP-response element sites in the proximal promoter region of Foxo1 gene.

Background Frequent exacerbations of allergic asthma lead to airway remodeling and a decrease in pulmonary function, producing morbidity. Cat dander is an aeroallergen associated with asthma risk. Objective We sought to elucidate the mechanism of cat dander–induced inflammation-remodeling. Methods We identified remodeling in mucosal samples from allergic asthma by using quantitative RT-PCR. We developed a model of aeroallergen-induced experimental asthma using repetitive cat dander extract exposure. We measured airway inflammation using immunofluorescence, leukocyte recruitment, and quantitative RT-PCR. Airway remodeling was measured by using histology, collagen content, myofibroblast numbers, and selected reaction monitoring. Inducible nuclear factor κB (NF-κB)–BRD4 interaction was measured by using a proximity ligation assay in situ. Results Enhanced mesenchymal signatures are observed in bronchial biopsy specimens from patients with allergic asthma. Cat dander induces innate inflammation through NF-κB signaling, followed by production of a profibrogenic mesenchymal transition in primary human small airway epithelial cells. The IκB kinase–NF-κB signaling pathway is required for mucosal inflammation-coupled airway remodeling and myofibroblast expansion in the mouse model of aeroallergen exposure. Cat dander induces NF-κB/RelA to complex with and activate BRD4, resulting in modifying the chromatin environment of inflammatory and fibrogenic genes through its atypical histone acetyltransferase activity. A novel small-molecule BRD4 inhibitor (ZL0454) disrupts BRD4 binding to the NF-κB–RNA polymerase II complex and inhibits its histone acetyltransferase activity. ZL0454 prevents epithelial mesenchymal transition, myofibroblast expansion, IgE sensitization, and fibrosis in airways of naive mice exposed to cat dander. Conclusions NF-κB–inducible BRD4 activity mediates cat dander–induced inflammation and remodeling. Therapeutic modulation of the NF-κB–BRD4 pathway affects allergen-induced inflammation, epithelial cell-state changes, extracellular matrix production, and expansion of the subepithelial myofibroblast population.

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.

Excessive ethanol drinking has deleterious effects on the brain. However, the effects of alcohol on microglia, the main mediator of the brain´s innate immune response remain poorly understood. On the other hand, the endocannabinoid system plays a fundamental role in regulating microglial reactivity and function. Here we studied the effects of acute ethanol exposure to murine BV2 microglial cells on N-acyl phosphatidylethanolamine-phospholipase D (NAPE-PLD), a major synthesizing enzyme of anandamide and other N-acylethanolamines. We found that ethanol downregulated microglial NAPE-PLD expression by activating cAMP/PKA and ERK1/2. These signaling pathways converged on increased phosphorylation of CREB. Moreover, ethanol induced an increase in histone acetyltransferase activity which led to higher levels of acetylation of histone H3. Taken together, our results suggest that ethanol actions on microglial NAPE-PLD expression might involve epigenetic mechanisms. Fil: Correa, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: de Laurentiis, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina

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.

Objective— Heparanase is an endoglycosidase that specifically cleaves carbohydrate chains of heparan sulfate. We have recently reported that high fatty acid increased the nuclear content of endothelial heparanase. Here, we examined the mechanism and the consequences behind this nuclear translocation of heparanase. Methods and Results— Bovine coronary artery endothelial cells were grown to confluence and incubated with palmitic acid. Palmitic acid induced rapid nuclear accumulation of heparanase that was dependent on Bax activation and lysosome permeabilization. Heat shock protein 90 was an important mediator of palmitic acid-induced shuttling of heparanase to the nucleus. Nuclear heparanase promoted cleavage of heparan sulfate, a potent inhibitor of histone acetyltransferase activity and gene transcription. A TaqMan gene expression assay revealed an increase in genes related to glucose metabolism and inflammation. In addition, glycolysis was uncoupled from glucose oxidation, resulting in accumulation of lactate. Conclusion— The results presented in this study demonstrate that fatty acid can provoke lysosomal release of heparanase, its nuclear translocation, activation of genes controlling glucose metabolism, and accumulation of lactate. Given that lactate and inflammation have been implicated in the progression of atherosclerosis, our data may serve to reduce the associated cardiovascular complications seen during diabetes.

Curcumin is an inhibitor of p300 histone acetyltransferase activity, which is associated with the deterioration of heart failure. We reported that native curcumin, at a dosage of 50 mg/kg, prevented deterioration of the systolic function in rat models of heart failure. To achieve more efficient oral pharmacological therapy against heart failure by curcumin, we have developed a novel drug delivery system (DDS) which markedly increases plasma curcumin levels. At the dosage of 0.5 mg/kg, DDS curcumin but not native curcumin restored left ventricular fractional shortening in post-myocardial infarction rats. Thus, our DDS strategy will be applicable to the clinical setting in humans.

Recent demonstration of the histone acetyltransferase activity of the Clock gene greatly expanded the regulatory role of circadian clocks in gene transcription. Clock and its partner Bmal1 are responsible for the generation of circadian oscillations that are synchronized (entrained) to the external light cycle. Entraining light often produces long-lasting changes in the endogenous period called aftereffects. Aftereffects are light-dependent alterations in the speed of free-running rhythms that persist for several weeks upon termination of light exposure. How light causes such long-lasting changes is unknown. However, the persistent nature of circadian aftereffects in conjunction with the long-term effects of epigenetic modifications on development and various aspects of brain physiology prompted us to hypothesize that the histone acetyltransferase CLOCK was required for circadian aftereffects. The authors exposed Clock knockout mice to 25-hour light cycles and report that these mice retain the ability to display circadian aftereffects, indicating that Clock is dispensable for this form of circadian plasticity.

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.

While many naturally occurring polyphenols have been shown to have therapeutic benefits against myocardial injury following ischemia-reperfusion in various experimental models, our studies have demonstrated that synthetic flavonoids may also have potent cardiac cytoprotective actions. Together with the results reported in the literature, we suggest that synthetic polyphenols may be an ideal replacement for natural compounds in the development of myocardial protective drugs. Polyphenols exert myocardial protective effects via antioxidant activities, preservation of nitric oxide, antiinflammatory activities and modulation of matrix metalloproteinases. Recent studies have identified some novel mechanisms that may also contribute to polyphenol-induced myocardial protection, including prevention of mitochondrial dysfunction, pharmacological preconditioning, and modulation of the function of enzymes involved in epigenetic modifications such as histone acetyltransferases. In addition to the protective effects against acute myocardial injury, there has been experimental evidence showing that polyphenols may also modulate the development of cardiac hypertrophy, ventricular remodeling and fibrosis after myocardial infarction.

Transcription factor Jun dimerization protein 2 (JDP2) binds directly to histones and DNA, and inhibits p300-mediated acetylation of core histones and reconstituted nucleosomes that contain JDP2-recognition DNA sequences. The region of JDP2 that encompasses its histone-binding domain and DNA-binding region is essential to inhibit histone acetylation by histone acetyltransferases. Moreover, assays of nucleosome assembly in vitro demonstrate that JDP2 also has histone-chaperone activity. The mutation of the region responsible for inhibition of histone acetyltransferase activity within JDP2 eliminates repression of transcription from the c-jun promoter by JDP2, as well as JDP2-mediated inhibition of retinoic-acid-induced differentiation. Thus JDP2 plays a key role as a repressor of cell differentiation by regulating the expression of genes with an activator protein 1 (AP-1) site via inhibition of histone acetylation and/or assembly and disassembly of nucleosomes. Senescent cells show a series of alterations, including flatten and enlarged morphology, increase in nonspecific acidic β-galactosidase activity, chromatin condensation, and changes in gene expression patterns. The onset and maintenance of senescence are regulated by two tumor suppressors, p53 and retinoblastoma proteins. The expression of p53 and retinoblastoma proteins is regulated by two distinct proteins, p16 Ink4a and Arf, respectively, which are encoded by cdkn2a . JDP2 inhibits recruitment of the polycomb repressive complexes 1 and 2 (PRC-1 and PRC-2) to the promoter of the gene that encodes p16 Ink4a and inhibits the methylation of lysine 27 of histone H3 (H3K27). The PRCs associate with the p16 Ink4a / Arf locus in young proliferating cells and dissociate from it in senescent cells. Therefore, it seems that chromatin-remodeling factors that regulate association and dissociation of PRCs, and are controlled by JDP2, might play an important role in the senescence program. The molecular mechanisms that underlie the action of JDP2 in cellular aging and replicative senescence by mediating the dissociation of PRCs from the p16 Ink4a / Arf locus are discussed.

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.