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

Abstract

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.

Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor

“…Collectively, our findings indicate that CHARM can be engineered to silence itself in vivo after silencing its target. Additional optimization of self-silencing kinetics will further improve the balance between target repression and timely discontinuation of transgene expression, such as by modulating the number of CpG sites in the promoter ( 81 ). For instance, systems for small-molecule control of self-silencing, such as tamoxifen-induced nuclear localization of a synthetic ZF transcriptional repressor ( 66 ), could allow for more precise temporal control as well as compatibility with different DNA binding domains.…”

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“…However, this study also detected DNAm leading to the upregulation of genes enriched for methylation-sensitive transcriptional repressors in their promoters. Recent synthetic models modulating CpG density and methylation state support a strong correlation between silencing and high CpG content, but also indicate potential sequence resolution intricacies with certain CpGs disproportionally controlling silencing [ 90 ]. It has also been recognised that CGI promoters can influence transcription through a CGCG tetranucleotide motif associated with highly expressed genes that binds the BANP (BTG3-associated nuclear protein) TF [ 91 ].…”