Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner

Abstract

Background: Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems. Results: Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9. Conclusions: These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

Nanobody-mediated control of gene expression and epigenetic memory

“…2d; blue and purple line). These observations were consistent with the reactivation dynamics of KRAB in a different mammalian cell line (CHO cells) 37 and the role of DNA methyltransferases in mediating epigenetic memory [5][6][7]38 . Overall, these results demonstrate the first use of a nanobody that can mediate gene silencing and impart epigenetic memory.…”

“…Recruitment of the catalytic domain of DNMT3A at a gene locus can induce DNA methylation and stable gene repression 39,40 . However, because DNMT3A alone typically leads to slow transcriptional repression, it is common to combine it with other CRs to enhance its effects [5][6][7] . Realizing the potential of the antiDNMT1 nanobody in improving the gene repressive effects of KRAB, we wanted to test if combining the nanobody with the catalytic domain of DNMT3A would enhance it as well.…”

“…Previous works have also shown that combining other CRs, such as MeCP2 and DNMT3A, with KRAB can further improve its silencing strength and epigenetic memory. Adding MeCP2 increases silencing strength at certain endogenous genes 4 while adding DNMT3A can increase silencing duration after a short initial pulse of gene targeting [5][6][7] . Further transcriptional control via histone deacetylation 8 , acetylation 9 , methylation 10 and demethylation 11 by CRs fused with dCas9 has also been reported, highlighting the flexibility of these tools in imparting diverse epigenetic modifications.…”

Quantitative dialing of gene expression via precision targeting of KRAB repressor

“…S6). Notably, we observed only modest repressive activity for DNA methyltransferase domains 16,33 when combined with the DNA binding domain of TM18, PD02, or LG09 (not shown).…”

Chemical and Light Inducible Epigenome Editing

“…Targeting G9A H3K9 methyltransferase to the promoter of HER2 in HCT116 cells increased H3K9me3 but did not lead to the transcriptional repression of HER2 [51]. Further studies of combinational targeting with EZH2 and other epigenome modifiers to HER2 revealed that long-term epigenetic memory can be achieved but in a context-dependent manner [52]. Several other epigenome modifiers have also been targeted with similar approaches for editing histone tail PTMs, including DOT1L [53], PRDM9 [53], HDAC3 [54], EZH2 [49], SUV39H1 [51], and G9A [51].…”