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

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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.