Reversing transgene silencing via targeted chromatin editing
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Abstract
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 CHO-K1 and human induced pluripotent stem cells to study the molecular interactions driving silencing and its reversal. After transient induction of either DNA methylation or H3K9me3, stable silencing was exclusively observed with both marks. Due to the positive feedback between DNA methylation and H3K9me3 and the relative low stability of H3K9me3, our model predicts that removing DNA methylation is sufficient for transgene reactivation. 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.
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Reversing transgene silencing via targeted chromatin editing
The combination of targeted chromatin editors and quantitative modeling provides a compelling framework for dissecting how DNA methylation and H3K9me3 cooperate to enforce transgene silencing. The work makes a strong case that DNA methylation is the primary heritable silencing mark in hiPSCs and that TET1-based demethylation can act as a modular anti-silencing tool.
I have a few questions about the CHO vs hiPSC comparison and the generality of the proposed feedback loop:
The key contrast hinges on KRAB installing H3K9me3 with or without subsequent DNA methylation. It’s hard to tell whether the absence of a feedback loop in CHO reflects a cell-type effect, a species effect, or CHO-specific epigenetic drift. Have you tested KRAB-mediated silencing and methylation in any …
Reversing transgene silencing via targeted chromatin editing
The combination of targeted chromatin editors and quantitative modeling provides a compelling framework for dissecting how DNA methylation and H3K9me3 cooperate to enforce transgene silencing. The work makes a strong case that DNA methylation is the primary heritable silencing mark in hiPSCs and that TET1-based demethylation can act as a modular anti-silencing tool.
I have a few questions about the CHO vs hiPSC comparison and the generality of the proposed feedback loop:
The key contrast hinges on KRAB installing H3K9me3 with or without subsequent DNA methylation. It’s hard to tell whether the absence of a feedback loop in CHO reflects a cell-type effect, a species effect, or CHO-specific epigenetic drift. Have you tested KRAB-mediated silencing and methylation in any additional mammalian cell types (such as human somatic lines or another rodent line), or mined existing datasets to see whether H3K9me3–DNMT3A coupling is generally weaker outside pluripotent cells?
You show that DNMT3A recruitment produces stable silencing and that TET1 can collapse both DNA methylation and H3K9me3 at the reporter. This is a powerful tool regardless, but it would be interesting to know whether the same bistable logic applies to endogenous loci. Have you examined KRAB- or DNMT3A-targeted repression at endogenous promoters, or compared H3K9me3 and DNA methylation dynamics at native loci in the same cells?
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