Non-canonical function of histone methyltransferase G9a in the translational regulation of chronic inflammation

We report a novel translation-regulatory function of G9a, a histone methyltransferase and well-understood transcriptional repressor, in promoting hyperinflammation and lymphopenia; two hallmarks of endotoxin tolerance (ET)-associated chronic inflammatory complications. Using multiple approaches, we demonstrate that G9a interacts with multiple translation regulators during ET, particularly the N6-methyladenosine (m6A) RNA methyltransferase METTL3, to co-upregulate expression of certain m6A-modified mRNAs that encode immune-checkpoint and anti-inflammatory proteins. Mechanistically, G9a promotes m6A methyltransferase activity of METTL3 at translational/post-translational level by regulating its expression, its methylation, and its cytosolic localization during ET. Additionally, from a broader view extended from the G9a-METTL3-m6A translation regulatory axis, our translatome proteomics approach identified numerous “G9a-translated” proteins that unite the networks associated with inflammation dysregulation, T cell dysfunction, and systemic cytokine response. In sum, we identified a previously unrecognized function of G9a in protein-specific translation that can be leveraged to treat ET-related chronic inflammatory diseases. Overall design: MeRIP-Seq/m6A-seq was performed using THP-1 cells (wildtype, G9a knockout, METTL3 knockout) under different inflammatory conditions (N, NL, TL) to identify G9a/METTL3-corregulated transcripts showing differential m6A modification.

我们报道了组蛋白甲基转移酶、经典转录抑制因子G9a的一种新型翻译调控功能：其可促进过度炎症与淋巴细胞减少——这两种均为内毒素耐受（endotoxin tolerance，ET）相关慢性炎症并发症的标志性特征。本研究通过多种实验方法证实，在内毒素耐受进程中，G9a可与多种翻译调控因子相互作用，尤其是N6-甲基腺苷（m6A）RNA甲基转移酶METTL3，共同上调部分编码免疫检查点及抗炎蛋白的m6A修饰mRNA的表达。机制层面，在内毒素耐受过程中，G9a通过调控METTL3的表达、甲基化修饰及其胞质定位，在翻译/翻译后水平增强其m6A RNA甲基转移酶活性。此外，从G9a-METTL3-m6A翻译调控轴拓展的更广视角出发，我们通过翻译组蛋白质组学方法鉴定出大量"G9a调控翻译"的蛋白，这些蛋白关联炎症失调、T细胞功能异常及系统性细胞因子应答相关的调控网络。综上，本研究揭示了G9a此前未被认知的蛋白质特异性翻译调控功能，该功能可作为治疗内毒素耐受相关慢性炎症性疾病的潜在靶点。实验整体设计：在不同炎症条件（N、NL、TL）下，使用THP-1细胞（野生型、G9a基因敲除型、METTL3基因敲除型）进行MeRIP-Seq/m6A测序，以筛选受G9a/METTL3共同调控且存在差异m6A修饰的转录本。