Phanstiel Doug, Brumbaugh Justin, Berggren W Travis, Conard Kevin, Feng Xuezhu, Levenstein Mark E, McAlister Graeme C, Thomson James A, Coon Joshua J
Department of Chemistry, Integrated Program in Biochemistry, Genome Center of Wisconsin, University of Wisconsin, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4093-8. doi: 10.1073/pnas.0710515105. Epub 2008 Mar 7.
Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.
通过染色质进行的表观遗传调控被认为在多能性的建立和维持中起着关键作用。传统上，基于抗体的技术用于探测组蛋白尾部存在的特定翻译后修饰（PTM），但这些方法通常无法揭示单个组蛋白尾部上多种修饰的存在（组合密码）。在这里，我们描述了一种基于色谱和质谱的用于发现和定量组蛋白组合密码的技术。我们应用这种方法来破译来自人类胚胎干细胞（ES）的组蛋白H4尾部的74种离散组合密码。最后，我们对这些密码的丰度进行了定量，以揭示人类ES细胞分化过程中甲基化和乙酰化模式的显著变化。例如，仅在存在H4K20二甲基化的情况下才观察到H4R3甲基化；这种上下文特异性模式体现了该技术的强大之处。
