Decoding gene responsiveness to synthetic chromatin reader-actuators with multi-modal epigenomic profiling.

Cell identity is regulated by chromatin states that encode gene regulatory memory and shape responses to new inputs. To investigate how chromatin context influences inducibility in differentiated cells, we employed engineered synthetic reader-actuators (SRAs), fusion proteins containing the polycomb chromodomain (PCD) that binds H3K27me3. In MCF7 breast cancer cells, we mapped PCD-fusion occupancy by ChIP-seq and used RNA-seq to identify temporally resolved gene activation patterns. ChIP-seq profiling and machine learning models (MLM) demonstrated that PCD-fusion binding was predicted primarily by the absence of H3K4me3 or H3K9ac enrichment, suggesting selective accessibility at enhancers and chromatin transition zones. Among genes with SRA-enriched enhancers, the SRA-induced subset was distinguished by promoter features including bivalent histone modifications and enrichment of transcription factors NEUROD1 and MTA1. Collectively, our results demonstrate that SRA responsiveness depends on a specific chromatin signature beyond H3K27me3 alone. This study demonstrates the power of SRAs to dissect inducible chromatin features in their native genomic context, and suggests that epigenetically repressed regions in differentiated cells can retain regulatory plasticity.