DNMT3A-dependent DNA methylation shapes the endothelial enhancer landscape [RNA-seq]

DNA methylation plays a fundamental role in regulating transcription during development, cell differentiation, and maintenance of cellular identity. However, the functional role of DNA methylation in the regulation of endothelial cell (EC) transcription during state transition, meaning the switch from an angiogenic to a quiescent cell state, has not been determined. Here, we conducted a DNA methylome analysis over a longitudinal postnatal timeline of EC in the murine pulmonary vasculature, revealing two significant observations. First, prominent alterations in DNA methylation patterns occurred during the transition from angiogenic to quiescent EC. Second, once a quiescent state is established, DNA methylation marks remain stable throughout further EC aging. These longitudinal differentially methylated regions correlated with endothelial gene expression and provided evidence for the recruitment of de novo DNA methyltransferase 3a (DNMT3A). Comprehensive loss-of-function studies in mice revealed that the absence of DNMT3A-dependent DNA methylation led to the loss of active enhancers, resulting in mild transcriptional changes, likely due to loss of active enhancer integrity. These results underline the importance of DNA methylation as a key epigenetic mechanism of EC function during state transition. Furthermore, we showed that DNMT3A-dependent DNA methylation appears to be involved in establishing the proper histone landscape required for accurate transcriptome regulation. Sequencing experiments were performed on FACS-sorted lung endothelial cells isolated from Dnmt3a KO and WT mice.DNA甲基化在发育进程、细胞分化及细胞身份维持过程中，对转录调控发挥着基础性作用。然而，DNA甲基化在内皮细胞（endothelial cell, EC）状态转换——即从血管生成态向静息细胞状态的切换——过程中，对内皮细胞转录的调控功能尚未明确。本研究针对小鼠肺血管中的内皮细胞，开展了出生后纵向时间序列的DNA甲基化组分析，得到两项重要发现：其一，从血管生成态向静息态内皮细胞转换期间，DNA甲基化模式出现显著改变；其二，静息细胞状态建立后，DNA甲基化标记会在内皮细胞后续衰老过程中保持稳定。这些纵向差异甲基化区域与内皮细胞的基因表达存在关联，并为从头DNA甲基转移酶3a（de novo DNA methyltransferase 3a, DNMT3A）的招募提供了实验证据。小鼠体内的系统性功能缺失实验显示，缺失依赖DNMT3A的DNA甲基化会导致活性增强子丢失，进而引发轻度转录变化，这可能源于活性增强子完整性的丧失。上述结果凸显了DNA甲基化作为内皮细胞状态转换过程中关键表观遗传调控机制的重要性。此外，本研究证实，依赖DNMT3A的DNA甲基化似乎参与构建精准调控转录组所需的适宜组蛋白修饰格局。本研究对从Dnmt3a基因敲除（KO）与野生型（WT）小鼠中分离得到的、经荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分选的肺内皮细胞开展了测序实验。