Development of a ‘tool box’ for generating designer nucleosomes in high throughput fashion
von Henriette Mahler
Datum der mündl. Prüfung:2016-12-22
Betreuer:Prof. Dr. Wolfgang Fischle
Gutachter:Prof. Dr. Wolfgang Fischle
Gutachter:Prof. Dr. Henning Urlaub
EnglischPost-translational modifications of histones within nucleosomes regulate cell development, cell maintenance and cellular response to external factors. According to the histone code hypothesis, each biologic reaction is encoded by a specific set of histone PTMs. In order to decipher the histone code, we developed a platform in which ligation-ready nucleosomes can be decorated with histone tails carrying PTMs of choice in a histone type specific manner. In this study, we presented the development of ligation-ready nucleosomes and histone type specific ligation of histone tails to H3 and H2A. For H3, Sortase A mediated ligation was chosen as it has been shown before to function at the histone level. For H2A, protein trans-splicing with an optimized mini-split intein (Ssp DnaB M86) was used. Ligation ready nucleosome core particle were assembled with H3 and H2A carrying tags which al- lowed a type selective approach. This meant for H3, truncation of the first 32 amino acids and for H2A, N-terminal removal of the fist 18 amino acids and attachment of the split-intein C-terminal part. Using these ligation strategies for H3 and H2A we were able to generate a NCP library with 36 combinatoric modified H3 and 8 variations of H2A modifications, resulting in a library with a complexity of 288. However, for the development of binding and activity assays only a 36 member H3-NCP library was used. The interpretation of histone marks that lead eventually to the regulation of cell development, cell maintenance and cellular response is carried out by effector proteins that ‘read’, ‘write’ or ‘erase’ histone PTMs. In order to gain insight in the ‘reading’ and ‘writing’ properties in dependence of H3-NCP modifications, we present the stage of development of a binding assay with fluorescence readout in 96-well format. As a ‘model’ protein was used in here, the well characterized HP1β. In addition, we explored the H3-NCP modification dependent activity of the histone acetyltransferase GCN5 and the kinase Aurora B in an enzymatic activity assay with readout of incorporated radioisotopes. For the HP1-binding assay, we found specific recruitment of enhanced cyan fluorescent protein labeled HP1 (eCFP-HP1) by H3K9me2 over H3um-NCP in a proof of principle experiment. This was detected by in-well fluorescence measurement and verified by western blot analysis. Experiments on the whole H3-NCP library brought up the question: how can we increase signal intensities and the ratio of eCFP-HP1 specific binding to H3K9me2- over H3um-NCP? This has been addressed by optimization of experimental factors (buffer composition, incubation time, wash steps) and the use of brighter CFP variants. Despite the optimizations in several directions we have been unable to develop a stable and robust binding assay. The setup of an high-throughput screening of the impact of modified H3-NCP on the activity of GCN5 and Aurora B presents an additional use of the immobilized NCP-library. Noticeable, a stark increase in overall acetylation was observed in the presence of H3S28ph or H3K27me2S28ph containing H3-NCP. This suggests a crucial role of PTMs in fine tuning the enzymatic activity of GCN5. In contrast, for Aurora B we observed that pre-phosphorylated H3-NCPs on both phosphorylation sites (H3S10ph/S28ph) were not a target for Aurora B anymore. In addition, we found that NCPs unmodified at H3S10 were better substrates for Aurora B than H3-NCPs containing pre-modified H3S10ph. This highlights the power of this assay to detect enzyme inherent properties as a function of pre-modified NCPs.
Keywords: Sortase A; epigenetic; nucleosome; histones; enzymatic assay; fluorescence; post translational modification; protein trans-splicing; semi-synthetic; library