Synthetic Epigenetic Reader Engineering with an All-in-One Cell-Free Expression and Protein Interaction Microarray

Chromatin, the system of genomic DNA, RNA, and proteins that organize chromosomes, is misregulated in cancer and has therefore become the target of an emerging class of epigenetic cancer therapies. These treatments rely heavily on small compounds to interfere with chromatin protein activity, while the vast potential of protein engineering to modulate chromatin in cancer has barely been explored. Our vision is to engineer synthetic reader-actuators (SRAs), customizable chromatin proteins that “read” chromatin features and modulate tumor suppressor and oncogene expression. Our immediate goal is to design an SRA with enhanced binding affinity for a cancer associated histone post-transcriptional modification, H3K27me3, using variants of the polycomb chromodomain (PCD) from the protein CBX8. So far, we achieved a 2-fold increase in SRA affinity for H3K27me3 by using two tandem wildtype PCDs (S. Tekel 2017 ACS Syn. Biol.). To enhance the affinity of a single PCD module, we will screen a library of variants that carry mutations at five amino acid positions that coordinate binding with the histone H3 tail. We developed a Golden Gate cloning platform (J.H. Priode 2021 protocols.io) to rapidly build a library of SRA variants in a cell-free transcription-translation vector (GGDestX1-Amp). We observed 50% Golden Gate insertion efficiency in GGDestX1-Amp, with zero misassembled products, and cell-free expression from GGDestX1-Amp in liquid phase reactions. We have designed a platform called the chromatin nucleic acid-programmable protein array (ChNAPPA), to efficiently screen all 3072 SRA constructs. This platform prints SRA-encoding DNA onto poly-L-lysine coated glass slides, alongside H3K27me3 histone tail peptides. After cell-free expression, the high-affinity variants are captured by the histone tail “baits” within each microspot. High-affinity SRA candidates from ChNAPPA will be tested for gene-activating function, compared to our standard SRAs (1x and 2x wildtype PCD-VP64), at silenced tumor suppressor genes in cancer cells in vitro.