Methionine metabolism in epigenetics of pluripotency and the germ line

Abstract

Metabolism sustains living organisms by generating the necessary molecular building blocks from nutrients in the environment. Intermediates in metabolic reaction pathways serve as critical substrates and cofactors to modify chromatin, making cellular metabolism a fundamental aspect of genome regulation and cell identity. S-adenosylmethionine (SAM) is an important co-factor for RNA, DNA and histone methylation. SAM is linked to the immediate precursor methionine (Met) and the one-carbon (1C) metabolic network more broadly, whose flux can influence abundance of SAM availability and therefore overall activity of epigenetic enzymes. My thesis examines the influence of 1C and Met metabolism on the epigenetic and epitranscriptomic regulation of pluripotent mouse embryonic stem cells (mESCs) cultured in two interchangeable conditions, 2i and Serum/Lif (SL). Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and stable isotope labelling (SIL) approaches, I determined hitherto unreported metabolic features that underlie the distinct epigenetic profiles of mESCs in these two conditions, including regulation of sulphur metabolism and methyl flux from Met and SAM. Notably, by using dialysed serum to control for serum’s amino-acid rich content, I significantly revise the consensus that mESCs are especially dependent on Thr, demonstrating that mESCs are also highly dependent on Met to proliferate normally and maintain their epigenetic configuration. Culture environment containing low Met concentrations for mESCs elicited a global histone response characterised by generally reduced methylation and increased acetylation. In SL-cultured mESCs, Met depletion caused reduced protein levels of OCT4 and NANOG pluripotency factors, along with diminished DNA and N6-methyladenosine (m6A) RNA methylation, encouraging significant questions into the interplay of Met and epigenetic marks in regulating the wider pluripotency network. My results significantly add to current knowledge of mESC metabolism and epigenetics, and may also have implications for our understanding of developmental epigenetic processes. Specifically, this work will help inform our efforts to delineate the role of metabolism in germline epigenetic reprogramming of primordial germ cells, in which genome-wide DNA demethylation and chromatin remodelling is accompanied by dynamic expression of 1C-metabolic enzymes.