Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.
Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia.
Genome Biol. 2022 Jul 26;23(1):163. doi: 10.1186/s13059-022-02728-5.
Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed.
Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II.
These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.
胞嘧啶 DNA 甲基化广泛被描述为一种转录抑制标记，具有沉默启动子的能力。表观基因组工程技术可用于直接测试诱导的 DNA 甲基化对内源性启动子的影响；然而，其下游效应尚未得到全面评估。
在这里，我们使用工程化锌指-DNMT3A 融合蛋白在人类细胞中同时诱导数千个启动子的甲基化，使我们能够测试强制 DNA 甲基化对转录、染色质可及性、组蛋白修饰和融合蛋白去除后 DNA 甲基化持久性的影响。我们发现，DNA 甲基化对转录的反应具有高度的上下文特异性，包括缺乏抑制以及增加基因表达的情况，这似乎是由甲基敏感转录抑制因子的驱逐所驱动的。此外，我们发现一些调控网络可以覆盖 DNA 甲基化，并且启动子甲基化可以导致替代启动子的使用。锌指-DNMT3A 融合蛋白去除后，启动子和远端调控区域的 DNA 甲基化迅速被擦除，这一过程结合了被动和 TET 介导的去甲基化。最后，我们证明，诱导的 DNA 甲基化可以同时存在于具有活性组蛋白修饰 H3K4me3 的启动子核小体上，或与起始形式的 RNA 聚合酶 II 结合的 DNA 上。
这些发现对表观基因组工程具有重要意义，并表明启动子对 DNA 甲基化的反应比以前认为的更为复杂。
