Quantifying Histone Modifications Associated with Age using Mass Spectrometry

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DESCRIPTION (provided by applicant): Aging changes the balance of pathway networks of all the cells in the body. The activity of the genetic material is one of the most permanent and widespread cellular function. While DNA is very obviously important, some arguably equally important players are the epigenetic proteins which control what parts of the DNA are available and transcribing. One group of these epigenetic proteins is called histones, which can be modified by acetylation, methylation, and phosphorylation to name a few. These chemical modifications on the proteins signal changes in DNA organization, replication, and repair. To profile the modifications on histones, a traditionally non-quantitative but sensitive and precise method, mass spectrometry, is used. Deuteroacetylation, an isotopic labeling strategy, allows even more sensitive identification and also quantification of site-specific modifications. The proposed experiments have three parts. 1) To develop a complementary mass spectrometric method (electron transfer dissociation) to increase histone sequence coverage of all four histones. It is expected that this will increase coverage of interesting residues from approximately 60% to 95%. The sample is deuteroacetylated to completion, cut into analyzable fragments, then analyzed with an orbitrap high resolution mass spectrometer. 2) To profile which and how much of each modification is present for six ages of yeast grown in low glucose (calorie restricting) or high glucose (non-restricting) media. Calorie restriction (CR) has been shown to increase the average lifespan and maximum age of yeast, mice and now humans. Experiments will use genetically modified yeast which contain HIS-tagged histones that are easily purified using Ni-NTA chromatography. Triplicate biological replicates will be correlated to a yeast viability study (to monitor effective age). Preliminary results show slight with age, and drastic changes during calorie-restriction. Some residues indicating aging effects list amongst the residues featured in aging studies by western blotting, confirming the preliminary results. 3) To validate the abundance of one particular modification of biological interest in the CR and aging conditions using three strategies: an alternative quantitative mass spectrometric method (multiple reaction monitoring), yeast enzyme knock-out strains and western blotting to determine if the histone modification decreases and what the effect is on the aging profile, and two human cell types (HEK293 stable cell culture and fresh peripheral blood mononuclear cell from human blood) will be used to confirm the modification using the original mass spectrometric method. Endogenous histones will be purified by an acidic purification method. This exciting new research will provide a full picture of how histones are implicated in aging and calorie restrictio, paving a superior workflow to improve productivity of histone research and provide new targets for aging biology researchers to study. PUBLIC HEALTH RELEVANCE: Histones, the proteins which organize DNA in all living things, are thought to be involved in the mechanism of aging biology through chemical modification. A deeper understanding of the histone role in aging could lead to drug targets to extend health into old age. We propose to use a new mass spectrometric technique to analyze histone changes throughout yeast aging, in human cell culture, and in fresh human peripheral blood mononuclear cell.