Dipartimento di Biologia e Patologia Cellulare e Molecolare "L, Califano", Facoltà di Farmacia e Facoltà di Scienze Biotecnologiche, Università degli Studi di Napoli "Federico II", Via S, Pansini 5, Naples, 80131, Italy.
BMC Microbiol. 2010 Jun 14;10:172. doi: 10.1186/1471-2180-10-172.
The release of LPS by bacteria stimulates both immune and specific epithelial cell types to release inflammatory mediators. It is known that LPS induces the release of IL-8 by intestinal mucosal cells. Because it is now emerging that bacteria may induce alteration of epigenetic patterns in host cells, we have investigated whether LPS-induced IL-8 activation in human intestinal epithelial cells involves changes of histone modifications and/or DNA methylation at IL-8 gene regulatory region.
RT-PCR analysis showed that IL-8 mRNA levels rapidly increase after exposure of HT-29 cells to LPS. DNA demethylating agents had no effects on IL-8 expression, suggesting that DNA methylation was not involved in IL-8 gene regulation. Consistently we found that 5 CpG sites located around IL-8 transcription start site (-83, -7, +73, +119, +191) were unmethylated on both lower and upper strand either in LPS treated or in untreated HT-29 cells, as well as in normal intestinal mucosa.Conversely, pretreatment of HT-29 cells with deacetylase inhibitors strengthened the LPS-mediated IL-8 activation. Inhibitors of histone deacetylases could induce IL-8 mRNA expression also in the absence of LPS, suggesting that chromatin modifications could be involved in IL-8 gene regulation. Chromatin immunoprecipitation analyses showed that, concurrently with IL-8 activation, transient specific changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation occurred at IL-8 gene promoter during LPS stimulation. Changes of H3-acetyl, H3K4me2 and H3K9me2 levels occurred early, transiently and corresponded to transcriptional activity, while changes of H3K27me3 levels at IL-8 gene occurred later and were long lasting.
The results showed that specific chromatin modifications occurring at IL-8 gene, including histone H3 acetylation and methylation, mark LPS-mediated IL-8 activation in intestinal epithelial cells while it is unlikely that DNA methylation of IL-8 promoter is directly involved in IL-8 gene regulation in these cells.
细菌释放的 LPS 刺激免疫和特定上皮细胞类型释放炎症介质。已知 LPS 诱导肠道黏膜细胞释放 IL-8。由于现在出现细菌可能诱导宿主细胞表观遗传模式的改变，我们研究了 LPS 是否诱导人肠道上皮细胞中 IL-8 的激活涉及 IL-8 基因调控区组蛋白修饰和/或 DNA 甲基化的变化。
RT-PCR 分析显示，LPS 暴露后 HT-29 细胞中 IL-8 mRNA 水平迅速增加。DNA 去甲基化剂对 IL-8 表达没有影响，表明 DNA 甲基化不参与 IL-8 基因调控。同样，我们发现，LPS 处理或未处理的 HT-29 细胞以及正常肠黏膜中，IL-8 转录起始位点（-83、-7、+73、+119、+191）周围的 5 个 CpG 位点的上下游均未甲基化。相反，HT-29 细胞的去乙酰化酶抑制剂预处理增强了 LPS 介导的 IL-8 激活。组蛋白去乙酰化酶抑制剂也可在没有 LPS 的情况下诱导 IL-8 mRNA 表达，表明染色质修饰可能参与 IL-8 基因调控。染色质免疫沉淀分析显示，在 LPS 刺激期间，与 IL-8 激活同时，IL-8 基因启动子处的 H3 乙酰化和 H3K4、H3K9 和 H3K27 甲基化发生短暂的特异性变化。H3-乙酰化、H3K4me2 和 H3K9me2 水平的变化发生较早、短暂，与转录活性相对应，而 IL-8 基因 H3K27me3 水平的变化发生较晚且持续时间较长。
结果表明，发生在 IL-8 基因上的特定染色质修饰，包括组蛋白 H3 乙酰化和甲基化，标记了 LPS 介导的肠道上皮细胞中 IL-8 的激活，而 IL-8 启动子的 DNA 甲基化不太可能直接参与这些细胞中 IL-8 基因的调控。
