The troupe of “toys” we use to explore the world of histone methylation just gained a new friend thanks to a playful team of epigenetic engineers who have taken histone modification-reading chromodomains to “affinity” and beyond!
Researchers led by Gianluca Veggiani (Louisiana State University, USA) and Sachdev Sidhu (University of Waterloo, Canada) knew that histone-modification reader domains could overcome problems associated with antibodies (cost, affinity, specificity, and lot-to-lot variations); however, modest binding strengths had limited their widespread application. Chromodomains, which can function as histone modification reader domains, display differences in methyllysine binding preferences and represent a critical resource if we can improve binding strengths for targets such as H3K9me3 and H3K27me3.
Let’s now hear from Veggiani and Martyn et al. on how engineering takes chromodomains to “affinity” and beyond:
- Phage display and site-directed mutagenesis combine to identify critical residues that enhance the relatively weak affinity of the CBX7 chromodomain for H3K27me3
- While engineered CBX7 variants display a significantly higher affinity for H3K27me3 peptides, one variant displays a twenty-fold improvement
- Functional characterization of engineered CBX chromodomains in mouse embryonic stem cells demonstrates their utility as probes for live-cell imaging and genome-wide binding analysis
- Engineered high-affinity chromodomains maintain the binding specificities of wild-type counterparts but display stronger binding to H3K27me3 in living cells
- Biotin-mediated chromatin immunoprecipitation followed by sequencing (biotin ChIP–seq) reports enhanced and specific H3K27me3 binding (no H3K9me3 binding)
- The combination of strongly binding CBX proteins with repressive KRAB domains fused to dCas9 provides a modular strategy for developing potent CRISPR interference (CRISPRi) approaches to repress gene expression
- Fusing CBX chromodomains to a KRAB domain and dCas9 enables cooperative increases in repressive activity (KRAB domains recruit various chromatin silencers) to induce higher levels of both H3K9me3 and H3K27me3
- The repressive potency of CBX-KRAB-dCas9 fusions positively correlates with chromodomain affinity, with specific engineered transcriptional repressors displaying greater potency than current gold standards
These results take histone methylation research to “affinity” and beyond by highlighting the power of engineered chromodomains to analyze chromatin-associated protein interaction networks and create gene-silencing platforms.
For more on how engineered chromodomains infinitely improve epigenetics research, see Nature Communications, November 2022.