SIRT1 regulates methionine metabolism and pluripotency of mouse embrynoic stem cells (mESCs)

The goal of this project is to understand the role of SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, in methionine metabolism and pluripotency of mESCs. Our recent studies indicate that SIRT1 deficient mESCs are hypersensitive to methionine restriction-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction leads to marked decrease in methylation levels of histones in SIRT1 deficient mESCs. To understand the impact of histone methylation alterations on stem cell functions in SIRT1 KO mESCs, we examined the transcriptomes of SIRT1 WT and KO mESCs cultured in either complete M10 medium (com) or methionine restricted medium containing 6 uM methionine (6met) for 6, 24, and 72 hours by microarray analysis. 3 groups of WT and SIRT1 KO mESCs were cultured in the complete medium (com) or methioine restricted medium (6met) for 6, 24, or 72 hours. Cells were then harvested and total RNA was isolated using the Qiagen RNeasy mini-kit. Gene expression profiles were analyzed using Agilent Whole Mouse Genome 4x44 multiplex format oligo arrays (014868) (Agilent Technologies, Santa Clara, CA), following the Agilent 1-color microarray-based gene expression analysis protocol.