Linking site-specific loss of histone acetylation to repression of gene expression by the mycotoxin ochratoxin A
Linking site-specific loss of histone acetylation to repression of gene expression by the mycotoxin ochratoxin A
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DOI：
10.1007/s00204-017-2107-6
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发表时间：
2017-11
影响因子：
6.1
通讯作者：
Elisabeth Limbeck;J. Vanselow;Julian Hofmann;A. Schlosser;A. Mally
中科院分区：
文献类型：
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作者：
Elisabeth Limbeck;J. Vanselow;Julian Hofmann;A. Schlosser;A. Mally
赭曲霉毒素 A (OTA) 是一种强效肾癌物质，但其机制尚未完全阐明。体外和体内基因表达研究一致表明，基因表达下调是 OTA 的主要转录反应。基于特定组蛋白乙酰化标记在调节基因转录中的重要性以及我们最近发现 OTA 抑制组蛋白乙酰转移酶 (HAT)，导致组蛋白和非组蛋白乙酰化丧失，我们假设 OTA 介导的基因表达抑制可能与 HAT 抑制和组蛋白乙酰化丧失存在因果关系。在这项研究中，我们使用了一种新颖的质谱方法，利用未修饰的赖氨酸残基的化学 13C 乙酰化来量化翻译后乙酰化位点，以识别暴露于 OTA 的人肾上皮细胞 (HK-2) 中组蛋白乙酰化的位点特异性改变。这些结果表明，核心组蛋白的几乎所有赖氨酸残基均存在 OTA 介导的低乙酰化，包括 H3K9 和 H3K14 乙酰化的丧失，这是基因激活的标志。 ChIP-qPCR 用于建立 H3K9 或 H3K14 低乙酰化与 OTA 介导的所选基因（AMIGO2、CLASP2、CTNND1）下调之间可能的联系，证实了 OTA 介导的这些基因启动子区域的 H3K9 低乙酰化。通过 ChIP-Seq 鉴定的 OTA 介导的 H3K9 乙酰化全基因组变化与已发表的基因表达数据的综合分析进一步表明，在 OTA 响应基因中，近 80% 的低乙酰化基因被下调，从而证实了 H3K9 乙酰化状态与这些基因的基因表达之间的关联。然而，只有 7% 的 OTA 抑制基因显示启动子区域内 H3K9 乙酰化缺失。然而有趣的是，GO 分析和下调基因的功能富集（显示各自启动子区域 H3K9 乙酰化的丧失）揭示了参与转录调节的基因的富集，包括许多转录因子，预计这些转录因子将直接或间接调节 98% OTA 抑制基因的表达。因此，一组相当小但在转录调控中具有关键功能的基因中的组蛋白乙酰化变化可能会引发一系列事件，从而导致基因表达的全面抑制。总而言之，我们的数据提供了证据，证明 OTA 介导的 HAT 抑制导致 H3K9 乙酰化丧失与 OTA 抑制基因表达之间存在机制联系，从而影响对肿瘤发生至关重要的细胞过程。
Ochratoxin A (OTA) is a potent renal carcinogen but its mechanism has not been fully resolved. In vitro and in vivo gene expression studies consistently revealed down-regulation of gene expression as the predominant transcriptional response to OTA. Based on the importance of specific histone acetylation marks in regulating gene transcription and our recent finding that OTA inhibits histone acetyltransferases (HATs), leading to loss of acetylation of histones and non-histone proteins, we hypothesized that OTA-mediated repression of gene expression may be causally linked to HAT inhibition and loss of histone acetylation. In this study, we used a novel mass spectrometry approach employing chemical13C-acetylation of unmodified lysine residues for quantification of post-translational acetylation sites to identify site-specific alterations in histone acetylation in human kidney epithelial cells (HK-2) exposed to OTA. These results showed OTA-mediated hypoacetylation at almost all lysine residues of core histones, including loss of acetylation at H3K9 and H3K14, which are hallmarks of gene activation. ChIP-qPCR used to establish a possible link between H3K9 or H3K14 hypoacetylation and OTA-mediated down-regulation of selected genes (AMIGO2,CLASP2,CTNND1) confirmed OTA-mediated H3K9 hypoacetylation at promoter regions of these genes. Integrated analysis of OTA-mediated genome-wide changes in H3K9 acetylation identified by ChIP-Seq with published gene expression data further demonstrated that among OTA-responsive genes almost 80% of hypoacetylated genes were down-regulated, thus confirming an association between H3K9 acetylation status and gene expression of these genes. However, only 7% of OTA repressed genes showed loss of H3K9 acetylation within promoter regions. Interestingly, however, GO analysis and functional enrichment of down-regulated genes showing loss of H3K9 acetylation at their respective promoter regions revealed enrichment of genes involved in the regulation of transcription, including a number of transcription factors that are predicted to directly or indirectly regulate the expression of 98% of OTA repressed genes. Thus, it is possible that histone acetylation changes in a fairly small set of genes but with key function in transcriptional regulation may trigger a cascade of events that may lead to overall repression of gene expression. Taken together, our data provide evidence for a mechanistic link between loss of H3K9 acetylation as a consequence of OTA-mediated inhibition of HATs and repression of gene expression by OTA, thereby affecting cellular processes critical to tumorigenesis.