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

Abstract

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.

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

“…To investigate if the wheat germ cell-free nucleosome assembly platform is suitable for the assembly of A. thaliana chromatin with histone variants, we co-expressed twelve combinations of canonical histones and their respective variants (Table 1 ) in the presence of relaxed pBSK plasmid DNAs 31 – 33 . Previously, a slight endogenous chromatin assembly activity was reported in the wheat germ extract during the reconstitution of Drosophila chromatin 33 . As both wheat and Arabidopsis are plants, we assumed Arabidopsis chromatin could be reconstituted in the wheat germ-based co-expression chromatin assembly system.…”

“…The supercoiling assay was used for the evaluation of the assembly of twelve chromatin combinations. The chromatin assembly reaction was carried out accordingly to the previous studies 31 – 33 . Briefly, in vitro transcribed mRNAs coding for all four core histone proteins were mixed in optimized ratios and co-expressed in a single reaction in the presence of pBSK plasmid DNA and topoisomerase I (Fig.…”

“…To this end, we attempted to reconstitute twelve A. thaliana nucleosome combinations from canonical and variant histones using the wheat germ cell-free co-expression chromatin assembly system described in recent studies 31 – 33 . Among the twelve tested combinations, seven types of nucleosomes were successfully reconstituted.…”