Post-translationale Protein Modifikationen, Querregulierung und Auswirkungen auf Histone H3 Tail Struktur und Dynamik: Eine strukturelle und mechanistische Studie an intakten Nukleosomen mittels hochauflösender Kernresonanzspektroskopie.
Biochemie
Zusammenfassung der Projektergebnisse
The main aim of the project was to expand my studies on characterizing the structural and dynamic properties of histone tails (focusing mainly on histone H3) and their post-translational modifications within a system that resembles at best the native occurring chromatin fiber. For that, I assembled nucleosomal arrays containing isotope-labeled histone proteins and performed NMR analyses. In-vitro reconstituted histone-DNA complexes were analyzed using a set of biochemical and microscopical assays that verified the correct assembly of pure and saturated arrays, which consisted of periodically spaced nucleosomes. The initial measurement of nucleosomal arrays containing isotope-labeled histone H3 by NMR spectroscopy resulted in good quality spectra that contained signals from all the H3 tail residues, thus constituting this system adequate for further NMR characterization. Subsequent NMR spectra of nucleosomal arrays containing isotope-labeled each one of the rest of histones pinpointed different dynamic features for the histone tails. While H3 and H4 tails were highly dynamic within nucleosomal arrays, in a similar mode as within single nucleosomes, histone H2A and H2B tails were surprisingly much less mobile, probably because they establish transient interactions with neighboring nucleosomes. In summary, these results constitute nucleosomal arrays an ideal physiological system for the structural characterization of histone tails by NMR spectroscopy and additionally revealed a differential behavior for the histone tails, distinguishing them in two groups with opposite dynamic properties. The second aim of the project was to investigate the mechanism that controls CBP acetyltransferase activity using as a system nucleosomes that contain differentially isotope-labeled pairs of histones. Up to now, there are no conclusive data on this topic mainly due to technical problems on the production of sufficient amounts of isotope-labeled histone H4 to be used for nucleosome reconstitution.
Projektbezogene Publikationen (Auswahl)
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Differentially isotope-labeled nucleosomes to study asymmetric histone modification crosstalk by time-resolved NMR spectroscopy. Angew. Chem. Int. Ed. 55(29): 8262-5 (2016)
Liokatis, S, klinberg, R, Tan, S, Schwarzer, D
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Modulations of DNA contacts by linker histones and post-translational modifications determine the mobility of nucleosomal H3 tails. Molecular Cell 61(2): 247-59 (2016)
Stützer, A, Liokatis, S, Kiesel, A, Schwarzer, D, Sprangers, R, Söding, J, Selenko, P, Fischle, W
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Reconstitution of nucleosomes with differentially isotope-labeled sister histones. J. Vis. Exp. (121), e55349 (2017)
Liokatis, S
