Post-translational modifications (PTMs) on histone proteins play a central role in epigenetic regulation and profiling the output of these discrete modifications is imperative for diagnosing and treating dysregulation. Antibodies designed for detecting PTMs on chromatin are an instrumental reagent in epigenetics research, however challenges such as off-target response, cross reactivity and reproducibility of detection persist in the use of antibodies. Reader domains are an attractive alternative for PTM detection since they have evolved to be both sequence-selective and modification-specific binders of histone PTMs. The binding affinity of a typical reader domain is orders of magnitude weaker than a conventional antibody, so our initial goal was to address this limitation of readers as detection reagents by improving upon the natural binding interaction. Thus far, we have engineered reader domains, termed eReaders, that recognize H3K9me to have stronger binding affinities than their native counterparts while maintaining PTM-site selectivity. By targeting highly conserved binding motifs at the protein-protein interface, we identified multiple mutations that collectively enhanced the binding affinity of HP1a from <i>D. melanogaster</i> for H3K9me3 by twenty-fold. We demonstrated that these beneficial mutations also strengthened the affinity of a human homolog, CBX1, and this eReader exhibits the tightest reported affinity for H3K9me3 to date. These binding improvements translated to more sensitive detection of H3K9me in histone-interaction assays, and notably the eReaders outperformed a robust commercial antibody in nucleosome blots. Our results present a growing library of eReaders that provide both reliable and robust PTM detection, are a cost-effective tool, and offer more modularity than traditional antibodies, thereby advancing the biochemical toolbox for epigenetic research.

Engineering reader domains as detection reagents for epigenetic modifications

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.