Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

Abstract

Background Elaboration 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. Results We 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. Conclusions The 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.

Development of a cell-free protein synthesis system for practical use

“…54), 55) Very recently, a simple protocol to reconstitute chromatin using this bilayer wheat system was reported, in which the mRNAs encoding the four core histones and chromatin assembly factors were co-expressed in the presence of a closed circular DNA. 56) A flow diagram of our cell-free system, combining the elementary technologies described above, is shown in Fig. 6.…”

De novo reconstitution of chromatin using wheat germ cell‐free protein synthesis

“…The methods for cloning of genes from Drosophila melanogaster into the pEU expression plasmid vector, preparation of a cell-free extract from wheat embryos and the transcription and translation reactions via a membrane dialysis device (Slide-A-Lyzer, Product #8841; Thermo Scientific, Rockford, IL, USA) were described previously [30][31][32]. In vitro transcription was performed as previously described using the pEU vector carrying an individual gene [33].…”

“…D. melanogaster histone sequences with UniProt IDs of DmH2A (P84051), DmH2B (P02283), DmH3 (P02299) and DmH4 (P84040) were used [30]. Wheat Triticum aestivum histone sequences were also retrieved from UniProt, including TaH2A (A03AB6IP09), TaH2B (W5A7K6), TaH3 (W5GCP8) and TaH4 (A0A3B6U3I5).…”

“…Recently, we developed a novel reconstitution method using a wheat germ cell-free coexpression system and showed both human and Drosophila chromatins were assembled onto a circular DNA in the presence of mRNAs coding four core histones and appropriate chromatin assembly factors [30]. Both supercoiling assay and micrococcal nuclease (MNase) digestion were used to assess the reconstituted chromatins.…”

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

“…Figure 1 provides information on the basic steps for conducting protein expression experiments in this WG-CFPS. These conditions allow for direct expression of proteins in high-throughput experiments or also joint expression of several proteins in a single reaction, as shown, for example, for chromatin reconstruction experiments using premixed mRNAs for up to four core histones, three chromatin assembly factors, and histone H1 ( Okimune et al, 2020 ). The expression of eight proteins in a single reaction is an impressive achievement not possible in most cell-based systems.…”