Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density
Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here, we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.
Copyright and License
© 2022 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0.
This work is supported by the Defense Advanced Research Projects Agency under contract no. HR0011-17-2-0008, by the National Institutes of Health grant RO1 HD075605A, and by National Science Foundation grant EF-2021552 under subaward UWSC10142. M.B.E. is a Howard Hughes Medical Institute Investigator.
Y.M.: conceptualization, formal analysis, investigation, and writing; J.Z.: formal analysis and investigation; M.W.B.: conceptualization, supervision, and funding; M.B.E: conceptualization, supervision, writing, and funding.
Conflict of Interest
The authors declare the following competing financial interest(s): M.W.B. is a founder and employee of Primordium Labs.
Plasmids and their maps available for requests at Addgene (addgene.org/browse/article/28233817/). The key cell lines are available upon request. EM-Seq raw and processed data is deposited at Gene Expression Omnibus (GSE224403). Data and codes for analysis and generating figures are available at data.caltech (doi: 10.22002/ct5kt-cv878).