Pluripotent stem cells (PSCs) have distinct metabolic properties that support their metabolic and energetic needs and affect their stemness. In particular, high glycolysis is critical for the generation and maintenance of PSCs. However, it is unknown how PSCs maintain and acquire this metabolic signature. In this study, we found that core pluripotency factors regulate glycolysis directly by controlling the expression of glycolytic enzymes. Specifically, Oct4 directly governs Hk2 and Pkm2, which are important glycolytic enzymes that determine the rate of glycolytic flux. The overexpression of Hk2 and Pkm2 sustains high levels of glycolysis during embryonic stem cell (ESC) differentiation. Moreover, the maintenance of high glycolysis levels by Hk2 and Pkm2 overexpression hampers differentiation and preserves the pluripotency of ESCs in the absence of leukemia inhibitory factor. Overall, our study identifies a direct molecular connection between core pluripotency factors and ESC metabolic signatures and demonstrates the significance of metabolism in cell fate determination. Stem Cells 2015;33:2699–2711

Acetylation is the most studied histone acyl modification and has been recognized as a fundamental player in metabolic gene regulation, whereas other short-chain acyl modifications have only been recently identified, and little is known about their dynamics or molecular functions at the intersection of metabolism and epigenetic gene regulation. In this study, we aimed to understand the link between nonacetyl histone acyl modification, metabolic transcriptional regulation, and cellular adaptation. Using antibodies specific for butyrylated, propionylated, and crotonylated H3K23, we analyzed dynamic changes of H3K23 acylation upon various metabolic challenges. Here, we show that H3K23 modifications were highly responsive and reversibly regulated by nutrient availability. These modifications were commonly downregulated by the depletion of glucose and recovered based on glucose or fatty acid availability. Depletion of metabolic enzymes, namely, ATP citrate lyase, carnitine acetyltransferase, and acetyl-CoA synthetase, which are involved in Ac-CoA synthesis, resulted in global loss of H3K23 butyrylation, crotonylation, propionylation, and acetylation, with a profound impact on gene expression and cellular metabolic states. Our data indicate that Ac-CoA/CoA and central metabolic inputs are important for the maintenance of histone acylation. Additionally, genome-wide analysis revealed that acyl modifications are associated with gene activation. Our study shows that histone acylation acts as an immediate and reversible metabolic sensor enabling cellular adaptation to metabolic stress by reprogramming gene expression.

Myocyte enhancer factor 2 (MEF2) is a family of transcription factors that regulates many processes, including muscle differentiation. Due to its many target genes, MEF2D requires tight regulation of transcription activity over time and by location. Epigenetic modifiers have been suggested to regulate MEF2-dependent transcription via modifications to histones and MEF2. However, the modulation of MEF2 activity by lysine methylation, an important posttranslational modification that alters the activities of transcription factors, has not been studied. We report the reversible lysine methylation of MEF2D by G9a and LSD1 as a regulatory mechanism of MEF2D activity and skeletal muscle differentiation. G9a methylates lysine-267 of MEF2D and represses its transcriptional activity, but LSD1 counteracts it. This residue is highly conserved between MEF2 members in mammals. During myogenic differentiation of C2C12 mouse skeletal muscle cells, the methylation of MEF2D by G9a decreased, on which MEF2D-dependent myogenic genes were upregulated. We have also identified lysine-267 as a methylation/demethylation site and demonstrate that the lysine methylation state of MEF2D regulates its transcriptional activity and skeletal muscle cell differentiation.