Abstract
BackgroundElaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies.ResultsWe describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications.ConclusionsThe method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.
Highlights
Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology
Histones and chromatin assembly factors synthesized by wheat germ cell-free synthesis The Drosophila core histones DmH2A, DmH2B, DmH3, and DmH4, and chromatin assembly factors, dAcf1, dISWI, and dNAP1 were cloned into the pEU-E01-MCS expression vector from the original pET plasmids [26, 40]
In the total of 744 proteins identified in the wheat germ cell-free extract, the most abundant proteins were annotated as heat shock proteins, followed by the proteins that related to protein synthesis such as a translation elongation factor
Summary
Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies. The majority of the abovementioned approaches utilize unmodified/ purified/refolded four core histones with defined DNA sequences that possess nucleosome positioning capability, and the assembly was achieved by the salt gradient method [16,17,18]. Based on these determined structures, the influence of particular post-translational modification of histone tails on nucleosome has been widely studied. Similar approaches were taken place to understand the role of post-translational modifications of histone proteins on the chromatin structure and functions [21, 22]
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