Establishing a Chemical Tool Box for Modified Nucleosomes and its Application for Analysis of the Histone Code

核小体构成染色质的基本结构单位，由组蛋白蛋白组成，组蛋白由大约150个碱基对的DNA组成支架。染色质紧凑真核基因组，是调节基因活性的重要手段，它是由组蛋白N端尾部的大量翻译后修饰所介导的。组蛋白修饰和基因活性之间复杂的相互作用的确切性质尚不清楚，部分原因是缺乏大量产生具有特定修饰模式的核小体的技术。在这里，我们建议建立一个工具箱，用于修饰核小体，在组蛋白H3和H4上定义修饰模式。为此，我们计划生成带有截短组蛋白H3和H4的准备连接的核糖体，这些组蛋白可以通过化学选择性连接技术与相应的合成尾巴垂直连接。为了实现这一计划，我们将在固定化核小体的背景下，建立一种改进的索尔特酶介导的组蛋白H3连接和蛋白质反式剪接的方法，该方法基于H4的SSP DNAB内含子。应合成一个复杂性为512个修饰模式的核小体文库，并将其用于以组合方式探测一组修饰依赖的组蛋白结合蛋白。精选的修饰核小体应进一步用于蛋白质组水平上的相互作用研究。此外，我们计划使用连接就绪的核小体在不同的组蛋白位置安装不同的荧光团，并筛选FRET供体和受体对的理想位置，用于定量结合研究。

Nucleosomes constitute the basic structural unit of chromatin composed of histone proteins that form a scaffold with about 150 base pairs of DNA wrapped around. Chromatin compacts eukaryotic genomes and represents an important means for regulating gene activity which is mediated by a multitude of posttranslational modifications on the N-terminal tail regions of the histones. The exact nature of the complex cross-talk between histone modifications and gene activity remains unknown in part due to the lack of techniques for generating nucleosomes with defined modification patterns in large numbers. Here we propose establishing a tool box for modified nucleosomes with defined modification patterns on histones H3 and H4. To this end we plan to generate ligation-ready nuclosomes with truncated histones H3 and H4, which can be orthogonally ligated with the corresponding synthetic tails by chemoselective ligation techniques. To realize this plan we will establish an improved sortase-mediated ligation for histone H3 and protein trans-splicing based on the Ssp DnaB intein for H4 in the context of immobilized nucleosomes. A nucleosomal library with a complexity of 512 modification patterns should be synthesized and used for probing a set of modification-dependent histone binding proteins in a combinatorial type of fashion. Selected modified nucleosomes should be further used for interaction studies on a proteomic level. In addition we plan to use ligation-ready nucleosomes for installing various fluorophores at different histone sites and screen for ideal positioning of FRET donor and acceptor pairs for quantitative binding studies.