Effects of environmental and genetic factors on Sir2 activity and DNA damage repair
Abstract
Environmental and genetic factors influence epigenetic states and affect cellular processes. Environmental factors cause heritable changes in gene expression patterns by affecting regulators of epigenetic processes. The plant secondary metabolite dihydrocoumarin (DHC), which is produced in several botanicals and is commonly used as a food, tobacco, and cosmetic additive, inhibits the Sir2p/SIRT1 family of NAD+-dependent histone deacetylases. In S. cerevisiae, Sir2p maintains silent chromatin at the HM loci, telomeres, and rDNA, whereas in mammals, SIRT1 regulates gene expression and other cellular processes. DHC causes disassembly of silent chromatin leading to loss of silencing at reporter and endogenous genes. Examination of the bioavailability and digestive stability of DHC in vivo and in vitro revealed that DHC was unstable and converted to melilotic acid (MA), a less bioactive compound. DHC did not accumulate to high levels in most rat tissues and was cleared from the animals 6 hr after DHC was administered. MA was present in the blood plasma, interstitial fluid, and urine of the animal and DHC was not detected in these samples suggesting that the conversion of DHC to MA occurs rapidly in the animals and is potentially catalyzed by enzymes in the bloodstream. To confirm that lower concentration of DHC could inhibit Sir2p activity independent of DHC hydrolysis to MA, α-factor confrontation assays were performed in strains lacking the multidrug transporter PDR5. Genetic factors also influence epigenetic processes. Asf1p, a chromatin assembly factor required for H3 K56ac by Rtt109p, binds H3/H4 dimers. Deletion of ASF1 leads to defects in silencing and sensitivity to DNA damaging agents. These phenotypes are attributed to H3 K56ac as strains lacking RTT109 and H3 K56R mutants have similar phenotypes to asf1δ strains. To separate genetically the role of Asf1p in chromatin assembly and H3 K56ac from DNA damage repair and binding to Rad53p, mutations were made to the surface of histone H3 that is bound by Asf1p. These mutations resulted in phenotypes that mimicked an asf1δ in all phenotypes tested except for H3 K56ac. H3 K56ac remained acetylated in all mutants tested, indicating that the H3-Asf1p interaction was not disrupted or the requirement for interaction between Asf1p and H3 for H3 K56ac had been bypassed in these mutants. These results appear to challenge the current understanding in the field as these phenotypes have previously been attributed to loss of H3 K56ac. Together, these two studies demonstrate the effects of a dietary factor, DHC, and genetic factors, Asf1p and H3/H4, on epigenetic processes in the cell.
Degree
M.S.
Advisors
Kirchmaier, Purdue University.
Subject Area
Molecular biology|Plant biology|Genetics|Biochemistry
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