BACKGROUND Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility may in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. The epigenetic modifier enhancer of zeste 2 (Ezh2) is a histone H3K27 methyltransferase implicated to play a role in lipid metabolism and adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate. We used the environmental chemical tributyltin (TBT) as a positive control, as this chemical is known to act on lipid metabolism via EZH-mediated pathways in mammals. RESULTS Zebrafish embryos (0-5 days post-fertilization, dpf) exposed to non-toxic concentrations of PF-06726304 acetate (5 μM) and TBT (1 nM) exhibited increased lipid accumulation. Changes in chromatin were analyzed by the assay for transposase-accessible chromatin sequencing (ATAC-seq) at 50% epiboly (5.5 hpf). We observed 349 altered chromatin regions, predominantly located at H3K27me3 loci and mostly more open chromatin in the exposed samples. Genes associated to these loci were linked to metabolic pathways. In addition, a selection of genes involved in lipid homeostasis, adipogenesis and genes specifically targeted by PF-06726304 acetate via altered chromatin accessibility were differentially expressed after TBT and PF-06726304 acetate exposure at 5 dpf, but not at 50% epiboly stage. One gene, cebpa, did not show a change in chromatin, but did show a change in gene expression at 5 dpf. Interestingly, underlying H3K27me3 marks were significantly decreased at this locus at 50% epiboly. CONCLUSIONS Here, we show for the first time the applicability of ATAC-seq as a tool to investigate toxicological responses in zebrafish. Our analysis indicates that Ezh2 inhibition leads to a partial primed state of chromatin linked to metabolic pathways which results in gene expression changes later in development, leading to enhanced lipid accumulation. Although ATAC-seq seems promising, our in-depth assessment of the cebpa locus indicates that we need to consider underlying epigenetic marks as well.

中文翻译：

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抑制zeste 2增强子的甲基转移酶活性导致斑马鱼中脂质积累增加和染色质状态改变。

背景技术最近的研究表明，暴露于环境化学物质可能增加对发展成代谢性疾病的敏感性。这种敏感性可能部分是由表观遗传环境的变化引起的，从而影响基因表达并导致脂质代谢的变化。Zeste 2（Ezh2）的表观遗传修饰增强子是一种组蛋白H3K27甲基转移酶，与脂质代谢和脂肪形成有关。在这项研究中，我们使用斑马鱼（Danio rerio）来研究Ezh2对Ezh1 / 2抑制剂PF-06726304醋酸盐的发育暴露后对脂质代谢和染色质状态的作用。我们使用环境化学三丁基锡（TBT）作为阳性对照，因为已知该化学物质通过EZH介导的哺乳动物途径参与脂质代谢。结果暴露于无毒浓度的PF-06726304乙酸盐（5μM）和TBT（1 nM）的斑马鱼胚胎（受精后0-5天，dpf）表现出增加的脂质蓄积。通过测定转座酶可及的染色质测序（ATAC-seq），在表位为50％（5.5 hpf）的情况下分析了染色质的变化。我们观察到349个染色质区域发生变化，主要位于H3K27me3位点，而暴露样品中的染色质大多开放。与这些基因座相关的基因与代谢途径相关。此外，在TBT和PF-06726304醋酸盐暴露于5 dpf（而不是50％外延阶段）后，差异表达脂类稳态，脂肪形成和PF-06726304醋酸盐通过改变染色质可及性专门靶向的基因。cebpa一个基因没有显示出染色质的变化，但确实显示了5 dpf时基因表达的变化。有趣的是，在该位点外显性H3K27me3标记显着下降了50％。结论在这里，我们首次展示了ATAC-seq作为研究斑马鱼毒理反应的工具的适用性。我们的分析表明，Ezh2抑制导致与代谢途径相关的染色质的部分致敏状态，从而导致基因表达在发育后期发生变化，从而导致脂质积累增加。尽管ATAC-seq看起来很有希望，但我们对cebpa基因座的深入评估表明，我们也需要考虑潜在的表观遗传标记。我们首次展示了ATAC-seq作为研究斑马鱼毒理反应的工具的适用性。我们的分析表明，Ezh2抑制导致与代谢途径相关的染色质的部分致敏状态，从而导致基因表达在发育后期发生变化，从而导致脂质积累增加。尽管ATAC-seq看起来很有希望，但我们对cebpa基因座的深入评估表明，我们也需要考虑潜在的表观遗传标记。我们首次展示了ATAC-seq作为研究斑马鱼毒理反应的工具的适用性。我们的分析表明，Ezh2抑制导致与代谢途径相关的染色质的部分致敏状态，从而导致基因表达在发育后期发生变化，从而导致脂质积累增加。尽管ATAC-seq看起来很有希望，但我们对cebpa基因座的深入评估表明，我们也需要考虑潜在的表观遗传标记。