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，染色质由组蛋白组成，组蛋白形成约150个碱基对的DNA包裹的支架。染色质是真核生物基因组的重要组成部分，是调控基因活性的重要手段，而基因活性的调控是通过组蛋白N端尾区的大量翻译后修饰来实现的。组蛋白修饰和基因活性之间的复杂串扰的确切性质仍然未知，部分原因是缺乏大量产生具有定义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，修改核小体与组蛋白H3和H4上定义的修饰模式。为此，我们计划用截短的组蛋白H3和H4生成连接就绪的核小体，其可以通过化学选择性连接技术与相应的合成尾正交连接。为了实现这一计划，我们将建立一个改进的分选酶介导的连接组蛋白H3和蛋白质反式剪接的基础上Ssp DnaB内含肽H4的背景下固定的核小体。一个具有512个修饰模式的复杂性的核小体库应该被合成并用于以组合类型的方式探测一组修饰依赖性组蛋白结合蛋白。选定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点安装各种荧光团，并筛选用于定量结合研究的FRET供体和受体对的理想定位。

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.