El Gazzar Mohamed, Yoza Barbara K, Chen Xiaoping, Hu Jean, Hawkins Gregory A, McCall Charles E
Department of Internal Medicine, Section of Molecular Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.
J Biol Chem. 2008 Nov 21;283(47):32198-208. doi: 10.1074/jbc.M803446200. Epub 2008 Sep 22.
TNFalpha gene expression is silenced in the endotoxin tolerant phenotype that develops in blood leukocytes after the initial activation phase of severe systemic inflammation or sepsis. The silencing phase can be mimicked in vitro by LPS stimulation. We reported that the TNFalpha transcription is disrupted in endotoxin tolerant THP-1 human promonocyte due to changes in transcription factor binding and enrichment with histone H3 dimethylated on lysine 9 (H3K9). Here we show that the TNFalpha promoter is hypermethylated during endotoxin tolerance and that H3K9 methylation and DNA methylation interact to silence TNFalpha expression. Chromatin immunoprecipitation and RNA interference analysis demonstrated that, in tolerant cells, TNFalpha promoter is bound by the H3K9 histone methyltransferase G9a which dimethylates H3K9 and creates a platform for HP1 binding, leading to the recruitment of the DNA methyltransferase Dnmt3a/b and an increase in promoter CpG methylation. Knockdown of HP1 resulted in a decreased Dnmt3a/b binding, sustained G9a binding, and a modest increase in TNFalpha transcription, but had no effect on H3K9 dimethylation. In contrast, G9a knockdown-disrupted promoter silencing and restored TNFalpha transcription in tolerant cells. This correlated with a near loss of H3K9 dimethylation, a significant decrease in HP1 and Dnmt3a/b binding and promoter CpG methylation. Our results demonstrate a central role for G9a in this process and suggest that histone methylation and DNA methylation cooperatively interact via HP1 to silence TNFalpha expression during endotoxin tolerance and may have implication for proinflammatory gene silencing associated with severe systemic inflammation.
在严重全身炎症或脓毒症初始激活阶段后血液白细胞中形成的内毒素耐受表型中，肿瘤坏死因子α（TNFalpha）基因表达沉默。这种沉默阶段可在体外通过脂多糖（LPS）刺激模拟。我们报道过，在内毒素耐受的THP - 1人原单核细胞中，由于转录因子结合的变化以及赖氨酸9（H3K9）上二甲基化组蛋白H3的富集，TNFalpha转录受到破坏。在此我们表明，在内毒素耐受期间TNFalpha启动子发生高甲基化，并且H3K9甲基化和DNA甲基化相互作用使TNFalpha表达沉默。染色质免疫沉淀和RNA干扰分析表明，在耐受细胞中，TNFalpha启动子与H3K9组蛋白甲基转移酶G9a结合，该酶使H3K9二甲基化并为异染色质蛋白1（HP1）结合创造一个平台，导致DNA甲基转移酶Dnmt3a/b的募集以及启动子CpG甲基化增加。敲低HP1导致Dnmt3a/b结合减少、G9a结合持续以及TNFalpha转录适度增加，但对H3K9二甲基化没有影响。相反，敲低G9a破坏了启动子沉默并恢复了耐受细胞中的TNFalpha转录。这与H3K9二甲基化几乎丧失、HP1和Dnmt3a/b结合以及启动子CpG甲基化显著减少相关。我们的结果证明了G9a在此过程中的核心作用，并表明组蛋白甲基化和DNA甲基化通过HP1协同相互作用，在内毒素耐受期间使TNFalpha表达沉默，并且可能与严重全身炎症相关的促炎基因沉默有关。
