miR-143-3p represses leukemia cell proliferation by inhibiting KAT6A expression.

The present study is designed to investigate the expressions of microRNA-143-3p (miR-143-3p) and Lysine acetyltransferase 6A (KAT6A) in acute myeloid leukemia (AML) samples and AML cell lines and to explore the possible effects and underlying mechanisms of miR-143-3p on the proliferation of AML cells. The expressions of miR-143-3p and KAT6A in AML samples and cell lines were detected by RT-qPCR assay. CCK-8 and flow cytometry were performed to evaluate the role of KAT6A in viability of AML cells. EdU assay was performed to determine the effects of KAT6A on proliferation of AML cells. Western blot analysis was utilized to assess the impacts of KAT6A on proliferation-related protein expressions of AML cells. ELISA assay was adopted to illustrate the influence of KAT6A on inflammatory responses of AML cells. In addition, the relationship between KAT6A and miR-143-3p was predicted by ENCORI and miRWalk, and confirmed by dual-luciferase reporter assay. Moreover, the effects of KAT6A on the proliferation of AML cells mediated with miR-143-3p were carried out by rescue experiment. The expression of KAT6A was significantly upregulated, while miR-134-4p was downregulated both in the AML tissues and in AML cell lines. In addition, the silence of KAT6A significantly inhibited the viability of AML cells. Besides, KAT6A silencing notably suppressed the proliferation of AML cells and reduced the protein expressions of Ki-67 and PCNA. Knockdown of KAT6A notably decreased the expression levels of IL-1ß, TNF-α and IL-6, and increased the expression levels of TGF-ß and IL-10. Moreover, overexpression of miR-143-3p repressed viability and proliferation of AML cells and overexpression of KAT6A partially reversed the inhibitory effects of miR-143-3p mimic on viability and proliferation of AML cells. miR-143-3p/KAT6A played an essential role in the viability and proliferation of AML cells.

Pneumocystis jirovecii Rtt109, a novel drug target for Pneumocystis pneumonia in immunosuppressed humans.

Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii. Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii, which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti-Pneumocystis agents against human P. jirovecii. Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109, Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109-null yeast. Recombinant PjRtt109 is an active HAT in vitro, with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro. PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro. Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti-Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP.

Repurposing disulfiram, an alcohol-abuse drug, in neuroblastoma causes KAT2A downregulation and in vivo activity with a water/oil emulsion.

Neuroblastoma, the most common type of pediatric extracranial solid tumor, causes 10% of childhood cancer deaths. Despite intensive multimodal treatment, the outcomes of high-risk neuroblastoma remain poor. We urgently need to develop new therapies with safe long-term toxicity profiles for rapid testing in clinical trials. Drug repurposing is a promising approach to meet these needs. Here, we investigated disulfiram, a safe and successful chronic alcoholism treatment with known anticancer and epigenetic effects. Disulfiram efficiently induced cell cycle arrest and decreased the viability of six human neuroblastoma cell lines at half-maximal inhibitory concentrations up to 20 times lower than its peak clinical plasma level in patients treated for chronic alcoholism. Disulfiram shifted neuroblastoma transcriptome, decreasing MYCN levels and activating neuronal differentiation. Consistently, disulfiram significantly reduced the protein level of lysine acetyltransferase 2A (KAT2A), drastically reducing acetylation of its target residues on histone H3. To investigate disulfiram's anticancer effects in an in vivo model of high-risk neuroblastoma, we developed a disulfiram-loaded emulsion to deliver the highly liposoluble drug. Treatment with the emulsion significantly delayed neuroblastoma progression in mice. These results identify KAT2A as a novel target of disulfiram, which directly impacts neuroblastoma epigenetics and is a promising candidate for repurposing to treat pediatric neuroblastoma.

Paeonol attenuates isoflurane anesthesia-induced hippocampal neurotoxicity via modulation of JNK/ERK/P38MAPK pathway and regulates histone acetylation in neonatal rat.

Objective: Volatile anesthetic such as isoflurane causes widespread neurodegeneration in the developing animal brains and also induces cognitive impairments. Paeonol is a plant-derived phenolic compound possessing numerous bioactive properties. The study investigates the neuroprotective effects of paeonol against isoflurane-induced neurodegeneration and cognitive disturbances in neonatal rats.Methods: Paeonol (50, 100, and 150 mg/kg body weight/day) was given orally to separate groups of neonatal rats from postnatal day 3 (P3) to P21 and were exposed to isoflurane (0.75%; 6 h) on P7.Results: Neuroapoptosis following isoflurane exposure was remarkably reduced by paeonol. Isoflurane-induced elevated cleaved caspase-3, Bad, and Bax expression, were down-regulated on paeonol administration. Paeonol significantly enhanced expression of antiapoptotic proteins (Bcl-2, Bcl-xL, xIAP, c-IAP-1, c-IAP-2, and survivin) and improved acetylation of HK39 and HK412. The expression of histone deacetylases (HDACs)-HDAC2 and HDAC-3 were down-regulated. Isoflurane-induced activation of JNK/p38MAPK signaling and suppressed ERK signaling and were effectively regulated by paeonol. General behavior and freezing responses of the rats were improved. Results of the Morris Water Maze tests revealed improved learning and memory retention on paeonol treatment.Conclusions: Paeonol effectively inhibited neuroapoptosis and improved isoflurane-induced cognitive dysfunctions via regulating histone acetylation and JNK/ERK1/2/p38MAPK signaling pathways.

Bortezomib-mediated inhibition of steroid receptor coactivator-3 degradation leads to activated Akt.

PURPOSE: To assess the safety of administering bortezomib to patients undergoing a radical prostatectomy, to assess pathologic changes induced by bortezomib in prostate cancer specimen, and to verify alterations by the drug in proteasome protein targets. EXPERIMENTAL DESIGN: Bortezomib is a proteasome inhibitor that has shown activity in vitro and in vivo in prostate cancer. We performed a neoadjuvant clinical trial of bortezomib in men with prostate cancer at high risk of recurrence. The primary endpoints were to evaluate safety and biological activity. RESULTS: Bortezomib is generally safe in the preoperative setting. Antitumor activity was manifested by tumor cytopathic effect, drops in serum prostate-specific antigen in some patients, and increases in tumor apoptosis. This was associated with cytoplasmic entrapment of nuclear factor-kappaB. We found an unexpected increase in proliferation in treated tissues and in vitro. Bortezomib also increased SRC-3 levels and phosphorylated Akt, both in vitro and in treated prostate cancer tissues. Knockdown of SRC-3 blocked the increase in activated Akt in vitro. Combined treatment with bortezomib and the Akt inhibitor perifosine was more effective than either agent alone in vitro. CONCLUSION: These data suggest that combined therapies targeting the proteasome and the Akt pathway may have increased efficacy.

Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model.

OBJECTIVE: We examined the potential of tannic acid (TA) as a novel histone acetyltransferase inhibitor (HATi) and demonstrated that TA prevents non-alcoholic fatty liver disease (NAFLD) by inhibiting HAT activity. METHODS: The anti-HAT activity of TA was examined using HAT activity assays. An in vitro NAFLD model was generated by treating HepG2 cells with oleic and palmitic acids. Male C57BL/6J mice were fed a control diet (CD) or Western diet (WD) with or without supplementation with either 1% or 3% TA (w/w) for 12 weeks. Finally, the possibility of interacting p300 and TA was simulated. RESULTS: TA suppressed HAT activity both in vitro and in vivo. Interestingly, TA abrogated occupancy of p300 on the sterol regulatory element in the fatty acid synthase and ATP-citrate lyase promoters, eventually inducing hypoacetylation of H3K9 and H3K36. Furthermore, TA decreased acetylation at lysine residues 9 and 36 of histone H3 protein and that of total proteins. Consequently, TA decreased the mRNA expression of lipogenesis-related genes and attenuated lipid accumulation in vivo. We observed that NAFLD features, including body weight, liver mass, fat mass, and lipid profile in serum, were improved by TA supplementation in vivo. Finally, we demonstrated the possibility that TA directly binds to p300 through docking simulation between ligand and protein. CONCLUSIONS: Our findings demonstrate that TA, a novel HATi, has potential application for the prevention of NAFLD.

The pro-inflammatory cytokines, IL-1beta and TNF-alpha, inhibit intestinal alkaline phosphatase gene expression.

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.

Effects of Scriptaid on the Histone Acetylation, DNA Methylation and Development of Buffalo Somatic Cell Nuclear Transfer Embryos.

The present study was undertaken to examine the effect of Scriptaid treatment on histone acetylation, DNA methylation, expression of genes related to histone acetylation, and development of buffalo somatic cell nuclear transfer (SCNT) embryos. Treatment of buffalo SCNT embryos with 500 nM Scriptaid for 24 h resulted in a significant increase in the blastocyst formation rate (28.2% vs. 13.6%, p<0.05). Meanwhile, treatment of buffalo SCNT embryos with Scriptaid also resulted in higher acetylation levels of H3K18 and lower methylation levels of global DNA at the blastocyst stage, which was similar to fertilized counterparts. The expression levels of CBP, p300, HAT1, Dnmt1, and Dnmt3a in SCNT embryos treated with Scriptaid were significantly lower than the control group at the eight-cell stage (p<0.05), but the expression of HAT1 and Dnmt1a was higher than the control group at the blastocyst stage (p<0.05). When 96 blastocysts developed from Scriptaid-treated SCNT embryos were transferred into 48 recipients, 11 recipients (22.9%) became pregnant, whereas only one recipient (11.1%) became pregnant following transfer of 18 blastocysts developed from untreated SCNT embryos into nine recipients. These results indicate that treatment of buffalo SCNT embryos with Scriptaid can improve their developmental competence, and this action is mediated by resulting in a similar histone acetylation level and global DNA methylation level compared to in vitro-fertilized embryos through regulating the expression pattern of genes related to histone acetylation and DNA methylation.

Novel antioxidants protect mitochondria from the effects of oligomeric amyloid beta and contribute to the maintenance of epigenome function.

Alzheimer's disease is associated with metabolic deficits and reduced mitochondrial function, with the latter due to the effects of oligomeric amyloid beta peptide (AßO) on the respiratory chain. Recent evidence has demonstrated reduction of epigenetic markers, such as DNA methylation, in Alzheimer's disease. Here we demonstrate a link between metabolic and epigenetic deficits via reduction of mitochondrial function which alters the expression of mediators of epigenetic modifications. AßO-induced loss of mitochondrial function in differentiated neuronal cells was reversed using two novel antioxidants (1 and 2); both have been shown to mitigate the effects of reactive oxygen species (ROS), and compound 1 also restores adenosine triphosphate (ATP) levels. While both compounds were effective in reducing ROS, restoration of ATP levels was associated with a more robust response to AßO treatment. Our in vitro system recapitulates key aspects of data from Alzheimer's brain samples, the expression of epigenetic genes in which are also shown to be normalized by the novel analogues.

The histone acetylase activator pentadecylidenemalonate 1b rescues proliferation and differentiation in the human cardiac mesenchymal cells of type 2 diabetic patients.

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone.

Acetylation is a key modulator of genome accessibility through decondensation of the chromatin structure. The balance between acetylation and opposite deacetylation is, in fact, a prerequisite for several cell functions and differentiation. To find modulators of the histone acetyltransferase Gcn5p, we performed a phenotypic screening on a set of newly synthesized molecules derived from thiazole in budding yeast Saccharomyces cerevisiae. We selected compounds that induce growth inhibition in yeast strains deleted in genes encoding known histone acetyltransferases. A novel molecule CPTH2, cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone, was selected based on its inhibitory effect on the growth of a gcn5Delta strain. We demonstrated a specific chemical-genetic interaction between CPTH2 and HAT Gcn5p, indicating that CPTH2 inhibits the Gcn5p dependent functional network. CPTH2 inhibited an in vitro HAT reaction, which is reverted by increasing concentration of histone H3. In vivo, it decreased acetylation of bulk histone H3 at the specific H3-AcK14 site. On the whole, our results demonstrate that CPTH2 is a novel HAT inhibitor modulating Gcn5p network in vitro and in vivo.

A mechanism for coordinating chromatin modification and preinitiation complex assembly.

Transcription of eukaryotic genes within a chromatin environment requires the sequential recruitment of histone modification enzymes and the general transcription factors (GTFs) by activators. However, it is unknown how preinitiation complex assembly is coordinated with chromatin modification. Here, we show that the model activator GAL4-VP16 directs the ordered assembly of Mediator, histone acetyltransferases (HATs), and GTFs onto immobilized chromatin and naked DNA templates in vitro. Using purified proteins, we found that the Mediator regulates this assembly process by binding to p300 and TFIID. An acetyl-CoA-dependent catalytic switch causes p300 to acetylate chromatin and then dissociate. Dissociation of p300 enhances TFIID binding and active transcription. The dissociation is caused by an autoacetylation-induced conformational change in the catalytic domain of p300. We conclude that autoacetylation-induced dissociation of p300 acts as a catalytic switch, which allows TFIID binding and subsequent preinitiation complex assembly.