Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density

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Abstract
Methylation of cytosinesin CG dinucleotides (CpGs) within promotershas been shown to lead to gene silencing in mammals in natural contexts.Recently, engineered recruitment of methyltransferases (DNMTs) atspecific loci was shown to be sufficient to silence synthetic andendogenous gene expression through this mechanism. A critical parameterfor DNA methylation-based silencing is the distribution of CpGs withinthe target promoter. However, how the number or density of CpGs inthe target promoter affects the dynamics of silencing by DNMT recruitmenthas remained unclear. Here, we constructed a library of promoterswith systematically varying CpG content, and analyzed the rate ofsilencing in response to recruitment of DNMT. We observed a tightcorrelation between silencing rate and CpG content. Further, methylation-specificanalysis revealed a constant accumulation rate of methylation at thepromoter after DNMT recruitment. We identified a single CpG site betweenTATA box and transcription start site (TSS) that accounted for a substantialpart of the difference in silencing rates between promoters with differingCpG content, indicating that certain residues play disproportionateroles in controlling silencing. Together, these results provide alibrary of promoters for synthetic epigenetic and gene regulationapplications, as well as insights into the regulatory link betweenCpG content and silencing rate.
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Funding Information
  • Defense Advanced Research Projects Agency (HR0011-17-2-0008)
  • National Institutes of Health (RO1 HD075605A)
  • National Science Foundation (EF-2021552, UWSC10142)

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