Department of Agricultural and Biological Engineering, Purdue University Center for Cancer Research, 225 South University Street, West Lafayette, IN 47907, USA.
J Cell Sci. 2012 Jun 15;125(Pt 12):2954-64. doi: 10.1242/jcs.101592. Epub 2012 Mar 5.
Although distinct epigenetic marks correlate with different chromatin states, how they are integrated within single nucleosomes to generate combinatorial signals remains largely unknown. We report the successful implementation of single molecule tools constituting fluorescence correlation spectroscopy (FCS), pulse interleave excitation-based Förster resonance energy transfer (PIE-FRET) and fluorescence lifetime imaging-based FRET (FLIM-FRET) to elucidate the composition of single nucleosomes containing histone variant H2A.Z (Htz1p in yeast) in vitro and in vivo. We demonstrate that yeast nucleosomes containing Htz1p are primarily composed of H4 K12ac and H3 K4me3 but not H3 K36me3 and that these patterns are conserved in mammalian cells. Quantification of epigenetic modifications in nucleosomes will provide a new dimension to epigenetics research and lead to a better understanding of how these patterns contribute to the targeting of chromatin-binding proteins and chromatin structure during gene regulation.
尽管不同的表观遗传标记与不同的染色质状态相关，但它们如何在单个核小体中整合以产生组合信号在很大程度上仍然未知。我们报告了成功实施单分子工具，包括荧光相关光谱（FCS）、基于脉冲交错激发的Förster 共振能量转移（PIE-FRET）和基于荧光寿命成像的 FRET（FLIM-FRET），以阐明体外和体内含有组蛋白变体 H2A.Z（酵母中的 Htz1p）的单个核小体的组成。我们证明，含有 Htz1p 的酵母核小体主要由 H4 K12ac 和 H3 K4me3 组成，但不包含 H3 K36me3，并且这些模式在哺乳动物细胞中是保守的。核小体中表观遗传修饰的定量将为表观遗传学研究提供一个新的维度，并有助于更好地理解这些模式如何有助于在基因调控过程中靶向染色质结合蛋白和染色质结构。
