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Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones
Published on: March 26, 2017
Differentially Isotope-Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time-Resolved NMR
Stamatios Liokatis1, Rebecca Klingberg2, Song Tan3
1Department of Structural Biology, Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125, Berlin, Germany. liokatis@fmp-berlin.de.
Abstract:
Post-translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual "sister" histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope-labeled nucleosomes to study asymmetric histone modification crosstalk by time-resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.
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