Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, 95616, USA.
Department of Biochemistry and Molecular Medicine, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA.
Epigenetics Chromatin. 2019 May 3;12(1):26. doi: 10.1186/s13072-019-0275-8.
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.
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.
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.
重写表观基因组已成为精准医学中基因编辑的一种有前途的替代方法。在自然界中，表观遗传沉默会导致靶基因表达在多个有丝分裂分裂中完全减弱。然而，使用靶向系统难以以可预测的方式实现持续抑制。
在这里，我们报告说，持续的表观遗传记忆既需要一种 DNA 甲基转移酶（DNMT3A-dCas9），也需要一种组蛋白甲基转移酶（Ezh2-dCas9 或 KRAB-dCas9）。我们证明组蛋白甲基转移酶的需求可以是特定于基因座的。与 KRAB-dCas9 不同，Ezh2-dCas9 与 DNMT3A-dCas9 和 DNMT3L 的共靶向作用诱导了至少 50 天（约 57 个细胞分裂）的长期 HER2 抑制，并引发了向异染色质环境的表观遗传开关。H3K27 三甲基化和 DNA 甲基化的增加被稳定维持，并伴有 H3K27 乙酰化的持续丧失。有趣的是，用 KRAB-dCas9 替代 Ezh2-dCas9 可使一些靶基因（例如 SNURF）长期受到抑制，但不能使 HER2 受到抑制，HER2 短暂获得 H3K9me3 和 DNA 甲基化，随后丢失。在许多单个 CpG 位点发生了脱靶 DNA 高甲基化，但在单个启动子中很少发生多个 CpG 位点的高甲基化，这与在测试的脱靶基因座上未检测到转录的影响一致。相反，在 HER2 上观察到强烈的高甲基化。我们进一步证明，Ezh2-dCas9 完全依赖全长 DNMT3L 发挥最大活性，并且共靶向 DNMT3L 对于 Ezh2-dCas9 或 KRAB-dCas9 的持续抑制是足够的。
这些数据表明，靶向不同组合的组蛋白和 DNA 甲基转移酶对于在不同基因座实现最大抑制是必需的。在任何给定的细胞类型中，在任何给定的基因座上构建表观遗传记忆都需要对靶向工具进行精细调整。
