Sidoli Simone, Trefely Sophie, Garcia Benjamin A, Carrer Alessandro
Department of Biochemistry and Biophysics, Perelman School of Medicine, Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA.
Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2019;1928:125-147. doi: 10.1007/978-1-4939-9027-6_9.
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, falls 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.
乙酰化是组蛋白上一种高度丰富且动态的翻译后修饰（PTM），当存在于与染色质结合的组蛋白上时，它有助于DNA进行基因转录。中心代谢物乙酰辅酶A是乙酰转移酶的底物，乙酰转移酶催化蛋白质乙酰化。乙酰辅酶A是多种代谢途径中的必需中间体，细胞内乙酰辅酶A水平会根据细胞外营养物质的可利用性和细胞的代谢状态而波动。大多数特异性作用于组蛋白的组蛋白乙酰转移酶（HATs）的米氏常数（Km）处于细胞内乙酰辅酶A浓度范围内。因此，组蛋白乙酰化的整体水平常常受到乙酰辅酶A可利用性的限制。组蛋白乙酰化的这种代谢调控对于细胞增殖、分化及多种细胞功能都很重要。在癌症中，众多致癌信号事件会操控细胞代谢，最终导致细胞表观遗传状态的广泛重排。通过组蛋白乙酰化了解表观基因组的代谢控制对于阐明细胞感知、适应以及偶尔脱离营养波动和环境信号与基因表达之间关系的分子机制至关重要。特别是，靶向代谢调节因子甚至进行饮食干预以影响乙酰辅酶A的可利用性和组蛋白乙酰化是癌症治疗中一个很有前景的靶点。液相色谱-质谱联用（LC-MS）是定量蛋白质翻译后修饰和代谢物最准确的方法。在本章中，我们介绍了用于分析组蛋白乙酰化和乙酰辅酶A的最新方案。在进行LC-MS分析之前，先提取组蛋白并将其消化成短肽（4 - 20个氨基酸）。从细胞中提取乙酰辅酶A，并使用类似的基于质谱的方法进行分析。可以给模型系统提供同位素标记的底物，以研究乙酰辅酶A产生的代谢偏好以及组蛋白乙酰化的代谢依赖性和周转率。我们还给出了一个数据整合的例子，以关联乙酰辅酶A产生的变化与组蛋白乙酰化。
