Mikula Michal, Majewska Aneta, Ledwon Joanna Karolina, Dzwonek Artur, Ostrowski Jerzy
Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw 02-781, Poland.
Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 02-781, Poland.
Int J Mol Med. 2014 Dec;34(6):1647-54. doi: 10.3892/ijmm.2014.1958. Epub 2014 Oct 3.
Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (p<0.05). Unexpectedly, the levels of H3K4me3 and H3K36me3 marks, as well as Pol2 and Nf-κB recruitment, did not correspond with the increased expression of these two genes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.
肥胖会导致非酒精性脂肪性肝病（NAFLD）的发生，其特征是两种关键炎症介质的表达上调：肿瘤坏死因子（Tnfa）和单核细胞趋化蛋白1（Mcp1；也称为Ccl2）。然而，肥胖个体肝脏中这些基因的染色质组成尚未得到研究。在本研究中，为了确定肥胖介导的Tnfa和Ccl2基因的表观遗传变化，我们使用了高脂饮食（HFD）诱导的肥胖小鼠模型和多食性（ob/ob）小鼠。染色质免疫沉淀（ChIP）分析用于确定允许性组蛋白标记的丰度，即组蛋白H3赖氨酸9和18乙酰化（H3K9/K18Ac）、H3赖氨酸4三甲基化（H3K4me3）和H3赖氨酸36三甲基化（H3K36me3），并结合肝脏中聚合酶2 RNA（Pol2）和核因子（Nf）-κB的募集情况。此外，为了将肝脏组织来源的ChIP测量结果与Tnfa和Ccl2基因强大的体外转录反应相关联，我们使用脂多糖（LPS）处理诱导源自小鼠肝细胞的细胞系Hepa1-6细胞发生炎症反应。ChIP显示肥胖小鼠中Tnfa和Ccl2基因的H3K9/K18Ac增加，尽管差异仅在Tnfa基因上具有统计学意义（p<0.05）。出乎意料的是，H3K4me3和H3K36me3标记的水平，以及Pol2和Nf-κB的募集情况，与肥胖小鼠中这两个基因表达的增加并不对应。相比之下，用LPS急性处理Hepa1-6细胞显著增加了两个基因的H3K9/K18Ac标记，以及Pol2和Nf-κB的募集，而H3K4me3和H3K36me3标记的水平保持不变。这些结果表明，脂肪肝中Tnfa和Ccl2基因表达的增加在染色质水平上与组蛋白H3乙酰化水平的变化相对应。
