Angewandte Synthetische Biologie
Using genetically installed unnatural amino acids we study dynamic properties of proteins mainly in the context of chromatin. We employ UAA incorporation to produce chromatin templates and proteins carrying post-translational modifications and fluorescent dyes at defined positions. This, combined with standard bioorthogonal chemistry and evolved orthogonal ribosomes [1-3], hall eventually facilitate single molecule studies of chromatin dependent processes in vitro.
The incorporation of UAAs can also be used to produce proteins with site-specific post-translational modifications. During my postdoc I established the production of acetylated histones H2A, H2B and H3 [4, 5]. The same approach, however, failed to yield measurable amounts of modified histone H4. We have developed a strategy to stabilize this histone from degradation in E.coli, facilitating the production of milligram quantities of recombinant histone H4 containing single or multiple modifications . Using genetically encoded UV-crosslinker amino acids, we investigate the dynamics of protein-protein interactions in living yeast. With this approach we were able to identify a driving force of chromatin condensation and its regulation in mitosis. We could show that during early mitosis, phosphorylation of H3 T3 by Haspin and subsequently of H3 S10 by Aurora B kinase controls the recruitment of the deacetylase Hst2p to nucleosomes and the deacetylation of K16 on H4 (Figure 1). As a consequence, the tail of H4 starts interacting with neighbouring nucleosomes, promoting chromatin condensation. This work identifies a condensin independent driving force of chromosome condensation in mitosis and explains the controversy that condensation still occurs, at least to some extent, in their absence. In collaboration with the group of Yves Barral (ETH Zürich) we could further show that in the absence of this H4-tail mediated condensation mechanism, the ability of fitting the condensation state of chromosomes to spindle length, through "adaptive hypercondensation", is impaired [7, 8].
With the same crosslinking approach we have mapped the interaction network of the FACT complex in living yeast. The FACT complex is an essential histone chaperone involved in transcription, replication and repair processes. The complex consists of two major subunits making multiple contacts to nucleosomal histones and DNA. The precise mechanism of FACT function is under intense investigation and largely unclear. Our work has identified a role for the acidic C-terminus of the Pob3-subunit of this complex in histone binding and DNA replication .
1. Lammers, C., Hahn, L.E., and Neumann, H. (2014). Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes. Chembiochem 15, 1800-1804.
2. Neumann, H., Wang, K., Davis, L., Garcia-Alai, M., and Chin, J.W. (2010). Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441-444.
3. lved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat Biotechnol 25, 770-777.
4. Neumann, H., Hancock, S.M., Buning, R., Routh, A., Chapman, L., Somers, J., Owen-Hughes, T., van Noort, J., Rhodes, D., and Chin, J.W. (2009). A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation. Mol Cell 36, 153-163.
5. Neumann, H., Peak-Chew, S.Y., and Chin, J.W. (2008). Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat Chem Biol 4, 232-234.
6. Wilkins, B.J., Hahn, L.E., Heitmuller, S., Frauendorf, H., Valerius, O., Braus, G.H., and Neumann, H. (2015). Genetically encoding lysine modifications on histone H4. ACS Chem Biol 10, 939-944.
7. Kruitwagen, T., Denoth-Lippuner, A., Wilkins, B.J., Neumann, H., and Barral, Y. (2015). Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation. elife 4.
8. Wilkins, B.J., Rall, N.A., Ostwal, Y., Kruitwagen, T., Hiragami-Hamada, K., Winkler, M., Barral, Y., Fischle, W., and Neumann, H. (2014). A cascade of histone modifications induces chromatin condensation in mitosis. Science 343, 77-80.
9. Hoffmann, C., and Neumann, H. (2015). In Vivo Mapping of FACT-Histone Interactions Identifies a Role of Pob3 C-terminus in H2A-H2B Binding. ACS Chem Biol 10, 2753-2763.