Institute of Biochemistry, Faculty of Chemistry, Stuttgart University, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstraße 112, 70376 Stuttgart, Germany.
Epigenetics Chromatin. 2015 Mar 18;8:12. doi: 10.1186/s13072-015-0002-z. eCollection 2015.
DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter.
Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment.
The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.
DNA 甲基化和组蛋白 3 赖氨酸 9（H3K9）甲基化被认为是可以通过细胞分裂遗传的表观遗传标记。为了探索这些修饰的功能后果和稳定性，我们使用与锌指蛋白融合的 DNA 或 H3K9 甲基转移酶的催化结构域，靶向安装血管内皮生长因子 A（VEGF-A）启动子中的 DNA 或 H3K9 甲基化，锌指蛋白结合 VEGF-A 启动子中的一个位点。
靶向 DNA 和 H3K9 甲基转移酶的表达导致 VEGF-A 启动子中 DNA 甲基化或 H3K9 二甲基化和三甲基化的密集沉积，以及 VEGF-A 基因表达的下调。我们没有观察到 DNA 甲基化和 H3K9 甲基化之间的正反馈。当靶向甲基转移酶从细胞中丢失时，表观遗传标记、染色质环境和基因表达水平恢复到原始状态，表明在安装后，这两种甲基化标记都没有稳定地传播。
DNA 或 H3K9 甲基化与基因表达之间的明显负相关表明这些标记直接参与转录控制。瞬时诱导的沉默染色质状态的缺乏维持表明，表观遗传信号的稳定性基于由几个分子标记组成的表观遗传网络。因此，为了稳定重编程，可能需要功能相关的表观遗传标记的多价沉积或更持久的触发刺激。
