Reversing transgene silencing via targeted chromatin editing

Advances in cell engineering and mammalian synthetic biology are enabling the development of genetically engineered cells to uncover biological principles and develop next-generation biotechnologies. However, epigenetic silencing of transgenes remains a major challenge in the field, particularly for human induced pluripotent stem cells (hiPSCs). Here, we employ targeted chromatin editing to study the dynamics of transgene silencing at integrated reporters in both CHO-K1 cells and hiPSCs. We first induce silencing by transiently recruiting writers of DNA methylation or histone 3 lysine 9 trimethylation (H3K9me3) to educate a chromatin modification model, which predicts DNA demethylation is sufficient for transgene reactivation. Accordingly, we demonstrate efficient and stable reactivation in both cell types through transient recruitment of a chromatin regulator that erases DNA methylation, irrespective of whether durable silencing was achieved with DNA methylation, H3K9me3 or triggered by endogenous cellular machinery. These results shed light on molecular mechanisms at play in transgene silencing and provide engineering tools for potent and specific reactivation in mammalian cells.