Teaching Synthetic Transcription Factors to Read an Epigenetic Code

We have developed a novel engineered protein to manipulate aberrant DNA-protein packaging (chromatin) in cancer cells. “PcTF” (Polycomb-based transcription factor) contains an N-terminal peptide motif “PCD” from the effector protein CBX8, which recognizes the histone modification signal H3K27me3. The C-terminal VP64 activation domain of PcTF reactivates epigenetically-silenced genes. This work represents a critical advance in the emerging field of epigenetic engineering by determining the function of a peptide module that reads chromatin marks. Other work that has analyzed formaldehyde-fixed chromatin suggests that CBX8 recognizes only a subset of H3K27me3 marks. In order to determine PCD activity in live cells, we used PcTF to activate H3K27me3-marked genes. We expressed PcTF from transfected plasmid DNA in three different human cancer-derived cell types (ATCC: U-2 OS/ bone, SK-N-SH/ brain, and K562/ blood). Quantitative sequencing of total messenger RNA (RNA-seq) was used to compare target-gene expression in PcTF-treated and untreated cancer-derived cells. In human cancer-derived cell lines, silenced H3K27me3-enriched genes (verified by chromatin immunoprecipitation) show higher levels of mRNA in PcTF-expressing cells compared to mock-transfected controls. We identified direct targets, genes with PcTF signals near their promoters, as well as hundreds of secondary targets. PcTF co-regulates clusters of genes within a subset of H3K27me3-associated regions, demonstrating that the CBX8 motif is selective. PcTF-activated genes verified by qRT-PCR include master transcriptional regulators such as HOX, RUNX3, CASZ1, and IRF8. These regulators and their targets are inhibitors of cancer cell growth in many reported studies. Thus, PcTF represents a potentially powerful new fusion-protein-based method for cancer treatment.