Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway.
Epigenetics Chromatin. 2020 Feb 12;13(1):5. doi: 10.1186/s13072-020-0329-y.
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.
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.
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.
最近的研究表明，接触环境化学物质可能会增加患代谢疾病的易感性。这种易感性部分可能是由于表观遗传景观的变化引起的，这些变化继而影响基因表达并导致脂质代谢发生变化。表观遗传修饰剂增强子的组蛋白 3 赖氨酸 27 甲基转移酶 2（Ezh2）被认为在脂质代谢和脂肪生成中发挥作用。在这项研究中，我们使用斑马鱼（Danio rerio）来研究在发育过程中暴露于 Ezh1/2 抑制剂 PF-06726304 乙酸盐后，Ezh2 对脂质代谢和染色质状态的作用。我们使用环境化学物质三丁基锡（TBT）作为阳性对照，因为这种化学物质已知在哺乳动物中通过 EZH 介导的途径作用于脂质代谢。
暴露于非毒性浓度的 PF-06726304 乙酸盐（5 μM）和 TBT（1 nM）的斑马鱼胚胎（受精后 0-5 天，dpf）表现出脂质积累增加。在 50%原肠胚形成（5.5 hpf）时，通过转座酶可及染色质测序（ATAC-seq）分析染色质变化。我们观察到 349 个改变的染色质区域，主要位于 H3K27me3 位点，并且在暴露的样本中主要是更多的开放染色质。与这些基因座相关的基因与代谢途径有关。此外，在 TBT 和 PF-06726304 乙酸盐暴露后 5 dpf 时，与脂质动态平衡、脂肪生成和特定于 PF-06726304 乙酸盐的通过改变染色质可及性靶向的基因相关的一组基因的表达发生了差异，但在 50%原肠胚形成阶段则没有。一个基因 cebpa 没有改变染色质，但在 5 dpf 时改变了基因表达。有趣的是，在这个基因座的基础 H3K27me3 标记在 50%原肠胚形成时显著减少。
在这里，我们首次展示了 ATAC-seq 作为一种工具来研究斑马鱼中毒性反应的适用性。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启动状态，从而导致发育后期基因表达的变化，导致脂质积累增加。尽管 ATAC-seq 似乎很有前景，但我们对 cebpa 基因座的深入评估表明，我们需要考虑潜在的表观遗传标记。
