In vitro co-expression chromatin assembly and remodeling platform for plant histone variants.

Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A. thaliana nucleosome combinations. Seven combinations were successfully reconstituted and confirmed by supercoiling and micrococcal nuclease (MNase) assays. The effect of the remodeling function of the CHR11-DDR4 complex on these seven combinations was evaluated based on the nucleosome repeat length and nucleosome spacing index obtained from the MNase ladders. Overall, the current study provides a novel method to elucidate the formation and function of a diverse range of nucleosomes in plants.

Histone chaperone-mediated co-expression assembly of tetrasomes and nucleosomes.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone-mediated assembly of both tetrasomes and nucleosomes on the well-defined Widom 601 positioning sequence using a co-expression/reconstitution wheat germ cell-free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell-free platform, our method is expected to be useful for downstream applications in the field of chromatin research.

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

DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell-free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP-1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone-modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.

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

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.