Reversing transgene silencing via targeted chromatin editing

Mammalian cell engineering offers the opportunity to uncover biological principles and develop next-generation biotechnologies. However, epigenetic silencing of transgenes hinders the control of gene expression in mammalian cells. Here, we use chromatin editing of an integrated reporter in both CHO-K1 and human induced pluripotent stem cells to dissect how histone H3 lysine 9 trimethylation (H3K9me3) and DNA methylation cooperate to maintain the silenced state and how this state can be reversed. After transient induction of either DNA methylation or H3K9me3, stable silencing was exclusively observed with both marks. Our chemical reaction model of chromatin modifications predicts that DNA methylation drives silencing maintenance. Accordingly, targeted DNA demethylation reactivated the reporter irrespective of whether silencing was achieved by inducing DNA methylation, H3K9me3, or by the endogenous cellular machinery. These results shed light on molecular mechanisms at play during silencing and provide engineering tools for potent and specific transgene reactivation in mammalian cells.