Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones

Abstract

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 D. 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 homolog, 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-*

Interactions With Histone H3 & Tools to Study Them

“…Alternatives, such as recombinant antibodies and purified histone modification interaction domains (HMIDs), are being developed and show promise (Kungulovski et al, 2014;Hattori and Koide, 2018). For example, a recently engineered HP1 chromodomain is reported to surpass antibodies in avidity when binding H3K9me3, without losing specificity (Albanese et al, 2020). Once validated, HMIDs can be expressed and purified at the required scale, eliminating lot variations associated with polyclonal antibodies.…”

Advances and Opportunities in Epigenetic Chemical Biology

“…[205] This has been achieved with both synthetic receptors [206][207][208][209] and engineered Chromodomains. [210,211] These synthetic receptors have proven useful as inhibitors, [207] antibody replacements [210] and fluorogenic sensors. [212,213] In some cases, all chemical artificial readers can approach recognition of Kme3 with an affinity similar to protein readers.…”

“…Significant effort has been expended in the chemical biology community to develop synthetic readers of lysine methylation [205] . This has been achieved with both synthetic receptors [206–209] and engineered Chromodomains [210,211] . These synthetic receptors have proven useful as inhibitors, [207] antibody replacements [210] and fluorogenic sensors [212,213] .…”

Selective Triazenation Reaction (STaR) of Secondary Amines for Tagging Monomethyl Lysine Post‐Translational Modifications

“…Moreover, they cannot be used to identify unknown Kme PTMs due to their high sequence specificity. Recently, small molecules and peptides mimicking chromodomains have been reported for identifying trimethyl lysine (Kme3) PTMs but tools to selectively detect Kme are still lacking [33, 34] . Another widely used approach for detecting Kme utilizes mass spectrometry (MS).…”

“…[30][31][32] Moreover,t hey cannot be used to identify unknown Kme PTMs due to their high sequence specificity.R ecently, small molecules and peptides mimicking chromodomains have been reported for identifying trimethyl lysine (Kme3) PTMs but tools to selectively detect Kme are still lacking. [33,34] Another widely used approach for detecting Kme utilizes mass spectrometry (MS). Although powerful but MS analysis is expensive,t ime-consuming and can be complicated by analytical challenges,including the low natural abundance of Kme PTMs in complex mixtures.Also,the change in the mass by monomethylation is identical to the substitutions of some amino acids thus leading to the false identification.…”