Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones

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. 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.
Here we 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, we 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.