The Flavoring Agent Dihydrocoumarin Reverses Epigenetic Silencing and Inhibits Sirtuin Deacetylases

Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, DHC inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases

Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomycescerevisiae

SWR1-Com, which is responsible for depositing H2A.Z into chromatin, shares four subunits with the NuA4 histone acetyltransferase complex. This overlap in composition led us to test whether H2A.Z was a substrate of NuA4 in vitro and in vivo. The N-terminal tail of H2A.Z was acetylated in vivo at multiple sites by a combination of NuA4 and SAGA. H2A.Z acetylation was also dependent on SWR1-Com, causing H2A.Z to be efficiently acetylated only when incorporated in chromatin. Unacetylatable H2A.Z mutants were, like wild-type H2A.Z, enriched at heterochromatin boundaries, but were unable to block spreading of heterochromatin. A mutant version of H2A.Z that could not be acetylated, in combination with a mutation in a nonessential gene in the NuA4 complex, caused a pronounced decrease in growth rate. This H2A.Z mutation was lethal in combination with a mutant version of histone H4 that could not be acetylated by NuA4. Taken together, these results show a role for H2A.Z acetylation in restricting silent chromatin, and reveal that acetylation of H2A.Z and H4 can contribute to a common function essential to life

The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases.

Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, DHC inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases

Eri1 regulates microRNA homeostasis and mouse lymphocyte development and antiviral function.

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3'-to-5' exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1(-/-) NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H(+) NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1(-/-) T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1(-/-) T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity

DHC Increases p53 Acetylation, Cytotoxicity, and Apoptosis in the TK6 Cell Line

<div><p>(A) DHC increases p53 lysine 373 and 382 acetylation in a dose-dependent manner in the TK6 cell line following a 24-h exposure period. The immunoblot is representative of three separate experiments.</p><p>(B) DHC increases cytotoxicity in a dose-dependent manner following a 24-h exposure to DHC. The average ± standard error for each of three experiments is shown.</p><p>(C) A 5-mM dose of DHC increases apoptosis at the 6-h time point in the TK6 cell line. A combination of annexin V fluorescein isothiocyanate and propidium iodide staining was used to discriminate among apoptotic (lower-right box) and necrotic (upper-right box) cells. Figures are representative of three separate experiments.</p><p>(D) DHC increases apoptosis in a dose-dependent manner in the TK6 cell line at the 6-h time point. The average ± standard error for three experiments is shown.</p></div

DHC Inhibits Human NAD⁺-Dependent Deacetylases In Vitro

<div><p>(A) Dose-dependent inhibition of SIRT1 (<i>y</i> = −19.98x + 96.431).</p><p>(B) Dose-dependent inhibition of SIRT2 (<i>y</i> = −20.79x + 101.34). Averages ± standard error from two separate experiments are shown.</p></div

Effect of DHC on Heterochromatin Silencing in S. cerevisiae at the HMR or HML Loci

<div><p>(A) DMSO negative control, 1 M nicotinamide positive control, and 50 mM DHC.</p><p>(B and C) DHC and splitomicin, respectively, cause dose-dependent increases in heterochromatin silencing and colony formation.</p></div