Integrated Analysis of Acetyl-CoA and Histone Modification via Mass Spectrometry to Investigate Metabolically Driven Acetylation

Abstract

Summary Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, fall within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4–20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation.

The Impact of One Carbon Metabolism on Histone Methylation

“…A new mass spectrometry method for histone posttranslational modification (PTM) analysis provides the possibility of high throughput screening of 200 PTMs in less than a minute (Sidoli et al, 2019a). Furthermore, methods that combine metabolomics and histone PTM analysis have already been developed for acetyl-CoA and histone acetylation (Sidoli et al, 2019b). These developments may not only help to broaden our view of how metabolism and epigenetic processes are connected but also open new pathways for pharmacological intervention in diseases.…”

Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear regulation

“…Acid extracted histone pellets were dissolved in 50 mM ammonium bicarbonate, pH 8.0, and histones were subjected to derivatization with as previously described (Sidoli et al, 2019b). Briefly, intact histones were mixed with 5 μL of deuterium-labeled propionic anhydride (propionic anhydride-d 10 ) rapidly followed by 14 μL of ammonium hydroxide (Sigma-Aldrich) to adjust to pH 8.0.…”

Cumulus cell acetyl-CoA metabolism from acetate is associated with maternal age but inconclusively with oocyte maturity

“…All tracing was conducted by laboratory analysts blinded to any sample characteristics using adaptations of previously developed methods [21]. CC metabolism was traced by centrifugation of the CC clump at 200 × g for 5 min at 25°C, removal of the supernatant, and then addition of the labeling media.…”