Endothelial transcriptome in response to pharmacological methyltransferase inhibition

The enzymatic activities of protein methyltransferases serve to write covalent modifications on histone and non-histone proteins in the control of gene transcription. Here, we describe gene expression changes in human endothelial cells caused by treatment with methyltransferase inhibitors 7,7'-carbonylbis (azanediyl) bis(4-hydroxynaphthalene-2 -sulfonic acid (AMI-1) and disodium-2-(2,4,5,7- tetrabromo-3-oxido-6-oxoxanthen-9-yl) benzoate trihydrate (AMI-5). Deep sequencing of mRNA indicated robust change on transcription following AMI-5 treatment compared with AMI-1. Functional annotation analysis revealed that both compounds suppress the expression of genes associated with translational regulation, suggesting arginine methylation by protein arginine methyltransferases (PRMTs) could be associated with regulation of this pathway. Interestingly, AMI-5 but not AMI-1 was found to decrease methylation of H3 histones at lysine 4 and down-regulate gene expression associated with interleukin-6 (IL-6) and activator protein-1 (AP-1) signaling pathways. These results imply that inhibition of protein methylation by AMI-1 and AMI-5 can differentially regulate specific pathways with potential to interrupt pathological signaling in the vascular endothelium. Overall design: HMEC-1 cells were treated with either 7,7'-carbonylbis (azanediyl) bis(4-hydroxynaphthalene-2 -sulfonic acid (AMI-1) or disodium-2-(2,4,5,7- tetrabromo-3-oxido-6-oxoxanthen-9-yl) benzoate trihydrate (AMI-5). These compounds were dissolved in DMSO and added to cells to make a final concentration of 100 µM. As a control, cells were exposed to DMSO only. Exposure time was 16 h.

蛋白质甲基转移酶的酶活作用是在组蛋白及非组蛋白上施加共价修饰，从而调控基因转录。本研究报道了甲基转移酶抑制剂7,7'-羰基双(氮杂二基)双(4-羟基萘-2-磺酸)（AMI-1）与三水合2-(2,4,5,7-四溴-3-氧代-6-氧代占吨-9-基)苯甲酸钠（AMI-5）处理人内皮细胞后引发的基因表达变化。对mRNA进行深度测序的结果显示，与AMI-1处理组相比，AMI-5处理后细胞的转录水平出现显著改变。功能注释分析表明，两种化合物均可抑制与翻译调控相关的基因表达，这提示蛋白质精氨酸甲基转移酶（protein arginine methyltransferases, PRMTs）介导的精氨酸甲基化可能参与该通路的调控。值得注意的是，仅AMI-5可降低组蛋白H3赖氨酸4位点的甲基化水平，并下调与白细胞介素-6（interleukin-6, IL-6）及激活蛋白-1（activator protein-1, AP-1）信号通路相关的基因表达。上述结果表明，AMI-1与AMI-5对蛋白质甲基化的抑制作用可差异性调控特定通路，具备干预血管内皮病理性信号转导的潜在能力。实验设计：将HMEC-1细胞分别用7,7'-羰基双(氮杂二基)双(4-羟基萘-2-磺酸)（AMI-1）或三水合2-(2,4,5,7-四溴-3-氧代-6-氧代占吨-9-基)苯甲酸钠（AMI-5）处理。两种化合物均以二甲基亚砜（dimethyl sulfoxide, DMSO）溶解，添加至细胞培养液中使其终浓度为100 μM；对照组细胞仅暴露于等量DMSO。处理时长为16小时。