Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering.

Abstract

Chromatin is the nucleoprotein complex that organizes genomic DNA in the nuclei of eukaryotic cells. Chromatin-modifying enzymes and chromatin-binding regulators generate chromatin states that affect DNA compaction, repair, gene expression, and ultimately cell phenotype. Many natural chromatin mediators contain subdomains that can be isolated and recombined to build synthetic regulators and probes. Engineered chromatin proteins make up a growing collection of new tools for cell engineering and can help deepen our understanding of the mechanism by which chromatin features, such as modifications of histones and DNA, contribute to the epigenetic states that govern DNA-templated processes. To support efficient exploration of the large combinatorial design space of synthetic chromatin proteins, we have developed a Golden Gate assembly method for one-step construction of protein-encoding recombinant DNA. A set of standard 2-amino acid linkers allows facile assembly of any combination of up to four protein modules, obviating the need to design different compatible overhangs to ligate different modules. Beginning with the identification of protein modules of interest, a synthetic chromatin protein can be built and expressed in vitro or in cells in under 2weeks.