Histone-Binding Domains as Modules for Custom Fusion Proteins

There is a growing interest in using peptide motifs from chromatin complexes to build new tools for probing and controlling epigenetic states within eukaryotic nuclei. Discoveries from basic research, including reverse genetics, biochemistry, and structural molecular biology, have illuminated the macromolecular interactions that organize chromatin. A central mechanism is the recognition of histone post-translational modifications (PTMs) by a family of proteins referred to as “readers.” Powerful molecular tools have emerged from this knowledge, enabling researchers to probe chromatin within living cells without the use of fixation and antibodies. For instance, histone PTM-binding domains (HBDs) have been expressed in-frame with fluorescent proteins to visualize the abundance and location of histone PTMs within cell nuclei. Our group has used HBDs to target synthetic transcriptional activators to sets of epigenetically repressed genes. Still, chromatin-associated proteins represent a vast design space that remains underexplored. To help fill this gap, we have developed a rapid prototyping platform to develop engineered fusion proteins that bind histone PTMs in vitro, and regulate genes near the target histone PTMs in living cells. We used cell-free production of fluorescently tagged fusion proteins to simplify the expression of libraries of fusion DNA constructs. Small volumes (less than 15 ul) of recombinantly-expressed, unpurified protein are sufficient for reproducible quantitation of histone PTM binding via enzyme-linked immunosorbent assays (ELISA), as well as qualitative analysis on histone peptide microspot arrays. We determined the gene-regulating activity HBD-bearing activators in living cells at a reporter transgene (HEK293 cells) and at endogenous loci that were enriched for a target histone PTM (H3K27me3 in K562 cells). Efforts to further simplify and miniaturize our design-build-test system are underway. We believe that this pipeline will be useful for synthetic biologists who are interested in designing novel proteins that can discriminate between the >100 known individual and combinatorial histone PTMs.