Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.
Radboud University, Faculty of Science, Department of Molecular Biology, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.
PLoS One. 2019 Jan 24;14(1):e0210217. doi: 10.1371/journal.pone.0210217. eCollection 2019.
Polycomb group (PcG) proteins are essential regulators of epigenetic gene silencing and development. The PcG protein enhancer of zeste homolog 2 (Ezh2) is a key component of the Polycomb Repressive Complex 2 and is responsible for placing the histone H3 lysine 27 trimethylation (H3K27me3) repressive mark on the genome through its methyltransferase domain. Ezh2 is highly conserved in vertebrates. We studied the role of ezh2 during development of zebrafish with the use of a mutant allele (ezh2(sa1199), R18STOP), which has a stop mutation in the second exon of the ezh2 gene. Two versions of the same line were used during this study. The first and original version of zygotic ezh2(sa1199) mutants unexpectedly retained ezh2 expression in brain, gut, branchial arches, and eyes at 3 days post-fertilization (dpf), as revealed by in-situ hybridization. Moreover, the expression pattern in homozygous mutants was identical to that of wild types, indicating that mutant ezh2 mRNA is not subject to nonsense mediated decay (NMD) as predicted. Both wild type and ezh2 mutant embryos presented edemas at 2 and 3 dpf. The line was renewed by selective breeding to counter select the non-specific phenotypes and survival was assessed. In contrast to earlier studies on ezh2 mutant zebrafish, ezh2(sa1199) mutants survived until adulthood. Interestingly, the ezh2 mRNA and Ezh2 protein were present during adulthood (70 dpf) in both wild type and ezh2(sa1199) mutant zebrafish. We conclude that the ezh2(sa1199) allele does not exhibit an ezh2 loss-of-function phenotype.
多梳抑制复合物（PcG）蛋白是表观遗传基因沉默和发育的重要调节剂。PcG 蛋白增强子的 zeste 同源物 2（Ezh2）是多梳抑制复合物 2 的关键组成部分，通过其甲基转移酶结构域负责在基因组上放置组蛋白 H3 赖氨酸 27 三甲基化（H3K27me3）抑制标记。Ezh2 在脊椎动物中高度保守。我们使用突变等位基因（ezh2（sa1199），R18STOP）研究了斑马鱼发育过程中 ezh2 的作用，该等位基因在 ezh2 基因的第二个外显子中具有终止突变。在这项研究中使用了相同品系的两个版本。第一个和原始版本的合子 ezh2（sa1199）突变体出人意料地在受精后 3 天（dpf）保留了大脑、肠道、鳃弓和眼睛中的 ezh2 表达，如原位杂交所揭示的。此外，纯合突变体的表达模式与野生型相同，表明突变的 ezh2 mRNA 不受无意义介导的衰变（NMD）的影响，如预测的那样。野生型和 ezh2 突变体胚胎在 2 和 3 dpf 时均出现水肿。通过选择性繁殖更新该系以对抗非特异性表型并评估存活。与早期的 ezh2 突变斑马鱼研究相比，ezh2（sa1199）突变体存活到成年。有趣的是，在野生型和 ezh2（sa1199）突变体斑马鱼中，成年期（70 dpf）均存在 ezh2 mRNA 和 Ezh2 蛋白。我们得出结论，ezh2（sa1199）等位基因不表现出 ezh2 功能丧失表型。
