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bioRxiv · 10.1101/2024.10.02.616304

An engineered chromatin protein with enhanced preferential binding of H3K27me3 over H3K9me3

Franklin, K. A.·Priode, J. H.·Steppe, P.·Haynes, K. A.
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Abstract

Histone post-translational modifications (PTMs) in chromatin modulate enhancer function, gene expression, and cellular differentiation. Pockets within chromatin reader-effector proteins bind specific histone PTMs, and have informed the design of artificial probes. Histone binding domains (HBDs) interact via multiple contacts that may support greater specificity than antibodies, but are difficult to study because of their weak affinities in vitro. We used a "Cell-Free Histone-Binding Immunoassay" (CHIA) where interactions between cell-free-expressed engineered HBD proteins and immobilized histone peptides are measured in an ELISA system. Previously reported K33E and Q9D variants of the chromobox 7 (CBX7) HBD bound with high affinity to both H3K27me3 and H3K9me3. However in CBX8, the K33E substitution enhanced binding to K27me3 with minimal K9me3 binding. Furthermore, hydrophobic substitutions in the CBX8 hydrophobic clasp (V10, L49) supported affinity and specificity for K27me3. We demonstrate the utility of a new CBX8 variant as a H3K27me3-specific live cell chromatin probe.

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Franklin, K. A., Priode, J. H., Steppe, P., Haynes, K. A.. 2024-10-02. An engineered chromatin protein with enhanced preferential binding of H3K27me3 over H3K9me3. https://doi.org/10.1101/2024.10.02.616304

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