Engineered eReader

Histone post-translational modifications, PTMs, are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics.

 

Magazine Cover

 

Wild type CBX1 reader protein can be engineered into a high affinity binder, eReader, for trimethyllysine on histone proteins. The eReader is able to recognize H3K9me3 in a whole nucleosome context, and performs better than wild type CBX1 and an anti-H3K9me3 antibody in relevant assays. Cover image by Lars Sahl

 

Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies, and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. In work published in ACS Chemical Biology researchers in the Waters Group, lead by Marcey Waters, featuring first author Katherine Albanese, describe engineered chromodomains from Drosophila melanogaster and humans that bind more tightly to H3K9 methylation, H3K9me, marks and result in the tightest reported reader–H3K9me interaction to date.

Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homologue, CBX1, resulting in an even tighter interaction with H3K9me3.

Furthermore, the group members behind this work show that these engineered readers, eReaders, increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-containing nucleosomes in vitro, demonstrating the utility of eReaders to complement antibodies in epigenetics research.