Medizinische Klinik für Kardiologie und Angiologie, Charité - Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Hypertension. 2012 Nov;60(5):1176-83. doi: 10.1161/HYPERTENSIONAHA.112.191098. Epub 2012 Sep 10.
The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) mediates trimethylation of lysine 27 in histone 3, which acts as a repressive epigenetic mark. Ezh2 is essential for maintaining pluripotency of stem cells, but information on its role in differentiated cells is sparse. Whole-genome mRNA expression arrays identified 964 genes that were regulated by >2-fold 72 hours after small interfering RNA-mediated silencing of Ezh2 in human umbilical vein endothelial cells. Among them, genes associated with the gene ontology terms cell communication and cell adhesion were significantly overrepresented, suggesting a functional role for Ezh2 in the regulation of angiogenesis. Indeed, adhesion, migration, and tube formation assays revealed significantly altered angiogenic properties of human umbilical vein endothelial cells after silencing of Ezh2. To identify direct target genes of Ezh2, we performed chromatin immunoprecipitation experiments followed by whole-genome promoter arrays (chromatin immunoprecipitation-on-chip) and identified 5585 genes associated with trimethylation of lysine 27 in histone 3. Comparative analysis with our mRNA expression data identified 276 genes that met our criteria for putative Ezh2 target genes, upregulation by >2-fold after Ezh2 silencing and association with trimethylation of lysine 27 in histone 3. Notably, we observed a striking overrepresentation of genes involved in wingless-type mouse mammary tumor virus integration site (WNT) signaling pathways. Epigenetic regulation of several of these genes by Ezh2 was specifically confirmed by polymerase chain reaction analysis of DNA enrichment after chromatin immunoprecipitation using an antibody specific for trimethylation of lysine 27 in histone 3. Combining mRNA expression arrays and chromatin immunoprecipitation-on-chip analysis, we identified 276 Ezh2 target genes in endothelial cells. Ezh2-dependent repression of genes involved in cell adhesion and communication contributes to the regulation of angiogenesis.
增强子结合锌指蛋白 2（Ezh2）是一种组蛋白甲基转移酶，可介导组蛋白 3 赖氨酸 27 的三甲基化，作为一种抑制性表观遗传标记。Ezh2 对于维持干细胞的多能性至关重要，但关于其在分化细胞中的作用的信息很少。全基因组 mRNA 表达谱分析鉴定出 964 个基因，这些基因在人脐静脉内皮细胞中通过小干扰 RNA 介导的 Ezh2 沉默 72 小时后，其表达水平被调控超过 2 倍。其中，与基因本体术语细胞通讯和细胞黏附相关的基因显著富集，这表明 Ezh2 在调节血管生成中具有功能作用。事实上，沉默 Ezh2 后，人脐静脉内皮细胞的黏附、迁移和管形成测定显示出明显改变的血管生成特性。为了鉴定 Ezh2 的直接靶基因，我们进行了染色质免疫沉淀实验，随后进行了全基因组启动子谱分析（染色质免疫沉淀芯片），并鉴定出与组蛋白 3 赖氨酸 27 三甲基化相关的 5585 个基因。与我们的 mRNA 表达数据的比较分析确定了 276 个基因，这些基因符合我们的假定 Ezh2 靶基因标准，即 Ezh2 沉默后上调超过 2 倍，并且与组蛋白 3 赖氨酸 27 三甲基化相关。值得注意的是，我们观察到参与 Wnt 信号通路的基因显著富集。通过使用特异性识别组蛋白 3 赖氨酸 27 三甲基化的抗体进行染色质免疫沉淀后 DNA 富集的聚合酶链反应分析，特异性地证实了这些基因中的几个基因受到 Ezh2 的表观遗传调控。通过 mRNA 表达谱分析和染色质免疫沉淀芯片分析，我们在内皮细胞中鉴定出 276 个 Ezh2 靶基因。Ezh2 依赖性抑制参与细胞黏附和通讯的基因有助于调节血管生成。
